Diagnostics for membranous nephropathy

ABSTRACT

The invention provides immunoassays for detecting serum auto-antibodies reactive against a phospholipase A2 receptor (PLA2R) and uses thereof for diagnosis and prognosis evaluation of idiopathic membranous nephropathy (MN).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S. patentapplication Ser. No. 13/006,864 filed on Jan. 14, 2011, which is acontinuation application of International Application No.PCT/US2009/051110 filed on Jul. 20, 2009, which designates the UnitedStates, and which claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/081,786 filed on Jul. 18, 2008, thecontents of each of which are incorporated herein by reference in theirentireties.

GOVERNMENT SUPPORT

This invention was made with Government support under contract No.DK067658 and DK30932 awarded by the National Institute of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 1, 2013, isnamed 701586-062708-C2_SequenceListing.txt and is 50,295 bytes in size.

BACKGROUND OF INVENTION

The nephrotic syndrome, characterized by edema and large amounts ofprotein in the urine, is a relatively common disorder of the kidney thathas many potential causes, including membranous nephropathy (MN), focaland segmental glomerulosclerosis, minimal change disease, diabeticnephropathy, membranoproliferative glomerulonephritis, as well as othercauses. Although there are non-specific treatments that ameliorate someof the signs and symptoms of the nephrotic condition, specific knowledgeof the underlying disease is usually necessary to guide definitivetreatment. When a patient with the nephrotic syndrome is initiallyevaluated as an outpatient or in the hospital, a panel of blood tests isusually ordered by the physician to look for potential causes that aredetectable by serology (for example, the presence of anti-nuclearantibodies (ANA) and/or anti-double stranded DNA antibodies in thecontext of typical findings in the urinary sediment would suggest lupusnephritis). There is currently no serologic test to identify MN, anddiagnosis relies exclusively on kidney biopsy, an invasive procedurerequiring overnight hospitalization in many institutions and one thatcan be complicated by major internal bleeding. MN can be caused by anumber of secondary factors, such as systemic lupus erythematosus,hepatitis B, or syphilis, and blood tests are routinely sent to look forthese causes (ANA, anti-hepatitis B antigens, rapid plasma reagin (RPR),respectively). However, in the United States, MN is more often primary,or idiopathic, in origin and as mentioned above, no blood test for thisform currently exists. Therefore, there is a need to identify theunderlying cause of MN and develop a simple, non-invasive test todiagnose MN and follow the response to treatment.

SUMMARY OF THE INVENTION

Embodiments of the invention are based on the discovery that theunderlying cause of idiopathic membranous nephropathy (MN) is thepresence of auto-antibodies against the M-type phospholipase A2 receptor(PLA2R) that is expressed in the kidney glomeruli. The auto-antibodiesare predominantly of the IgG4 subclass. In addition, anti-PLA2Rauto-antibodies of the subclass IgG1, IgG2, and IgG3 were also present.The sera of individuals having idiopathic MN have detectable amounts ofsuch auto-antibodies. Knowing that the presence of auto-antibodies isassociated with MN and that PLA2R is the target of these auto-antibodieshas enabled the development of a simple serological immunoassay fordiagnosing idiopathic MN. This detection of anti-PLA2R auto-antibodiesprovides a fast, accurate, cost-effective, safe and non-invasive methodof diagnosing idiopathic MN, compared to the current method of a kidneytissue biopsy.

Accordingly, in an embodiment, provided herein is a method of diagnosingMN in a subject, the method comprising detecting the presence ofantibodies that are reactive to a PLA2R, wherein the antibodies arefound in a sample from a subject. The antibodies can be detected by animmunoassay wherein an antibody-protein complex is formed. Theantibodies are found in the sample of the subject, e.g. serum. Thesubject is a human and the MN is idiopathic. Prior to the diagnosismethod, a kidney biopsy is not performed. Healthy individuals haveminimal or undetectable anti-PLA2R auto-antibodies by conventional ELISAor Western blots. Individuals with idiopathic MN have significant amountof detectable anti-PLA2R auto-antibodies, at least 10% more anti-PLA2Rauto-antibodies detected over that from a healthy non-MN individual orthe level obtained for a population of healthy non-MN individuals byconventional ELISA or Western blots as described herein. Moreover thelevels of auto-antibodies correspond with the clinical features of thedisease condition e.g. proteinuria and nephrotic syndrome. Patients inremission after effective treatment have minimal or undetectableanti-PLA2R auto-antibodies by conventional ELISA or Western blots. As anexamplary, by undetectable amount of anti-PLA2R auto-antibodies, itmeans that no visible band is observed in a Western Blot analysisperformed as described in Example 1, wherein human serum is diluted1:100 and used in blot assays described herein. In one embodiment, theamount of anti-PLA2R auto-antibodies in a healthy non-MN individual orthe average amount in a population of healthy non-MN individuals asdetermined by conventional ELISA or Western blot set forth in Example 1can be considered as the background, reference or the control level. Thecollected samples of serum from the healthy non-MN individuals arediluted 1:100 and used in Western blot assays. The intensity of thevisible band is quantified by densitometry. The densitometry intensitycan be calibrated with a range of known titer of anti-PLA2R antibodiesreacting with a fixed amount of antigen PLA2R. For example, the range ofknown antibody titer can be 0 μg/ml, 0.5 μg/ml, 1.0 μg/ml, 1.5 μg/ml,2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml, 5 μg/ml, 7.5 μg/ml, 10 μg/ml, and 15μg/ml and the fixed amount of PLA2R can be 0.5 μg on a blot. Bycomparing the densitometry intensity of a human sample with thecalibration curve, it is possible to estimate the titer of theanti-PLA2R in nthe sample. For the data collected for a population ofindividuals, the average value and one order of standard deviation iscomputed. Ideally, a population has about 25 healthy non-MN individuals,preferably more. The statistics, the average value and one order ofstandard deviation can be uploaded to the computer system and datastorage media. Patients having at least 10% more than this averageamount of anti-PLA2R auto-antibodies is likely to have MN, especially ifthe patient is also presents the clinical significant features of thedisease, e.g. proteinuria and nephrotic syndrome.

In one embodiment, the auto-antibodies in the sample are reactiveagainst the PLA2R that has been extracted from mammalian tissues orrecombinant mammalian PLA2R, e. g. the human or pig PLA2R. The samplefrom the subject can be a blood sample. In other embodiments, the sampleis a serum or plasma sample.

In one embodiment, the auto-antibodies are detected by a serologicalimmunoassay, such as an enzyme-linked immunosorbant assay or anephelometric immunoassay.

In an embodiment, provided herein is a method of prognosis evaluation ina subject being treated for membranous nephropathy, the methodcomprising: (a) determining at a first time point a level of antibodiesthat are reactive to a PLA2R, wherein the antibodies are found in asample from a subject; (b) determining at a second time point a level ofantibodies that are reactive to a PLA2R, wherein the second time pointis after the first time point; and (c) comparing the levels ofantibodies of the two time points, wherein a decrease in the level ofantibodies in the second time point compared to the first time pointindicates that the treatment is effective. The decrease is at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, 100% and all the percentagesbetween 10-100%. The level of the antibodies can be detected by animmunoassay wherein an antibody-protein complex is formed and thecomplex is detected. The antibodies are found in the sample of thesubject, e.g. serum. In an embodiment, the treatment is animmunosuppressive treatment. In one embodiment, the level of antibodiesat a later time point, e.g. the second time point, is between 95-100% ormore lower compared to the first time point, which is considered belowthe detection limit of the immunoassay, then the subject is in remissionfor MN. In one embodiment, below the detection limit of a Western blotis when no visible band is present when the assay is performed accordingto the method set forth in Example 1.

In an embodiment, provided herein is a method of prognosis evaluation ina subject for membranous nephropathy, the method comprising: (a)determining at a first time point a level of antibodies that arereactive to a PLA2R wherein the antibodies are found in a sample from asubject; and (b) determining at at least a second time point a level ofantibodies that are reactive to a PLA2R, wherein the second time pointis after the first time point; wherein when the in the level ofantibodies in the second time point decreases to below the detectionlimit indicates that there is spontaneous remission. The decrease is atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, 100% and all thepercentages between 10-100%. As discussed here, such decreases indicatethat the patient is getting better relative to the prior reading. Belowthe detection limit is when the level of antibodies is reduced tobetween 95-100% and beyond compared to the first time point. In oneembodiment, below the detection limit of a Western blot is when novisible band is observed when the assay is performed according to themethod set forth in Example 1.

In one embodiment, when the subject is being treated for MN and thelevel of antibodies in the second time point is below the detectionlimit of the immunoassay, then the subject is considered to be inremission for MN.

In an embodiment, provided herein is a method of prognosis evaluation ina subject for membranous nephropathy, the method comprising: (a)determining at a first time point a level of antibodies that arereactive to a PLA2R, wherein the antibodies are found in a sample from asubject; (b) determining at at least a second time point a level ofantibodies that are reactive to a PLA2R, wherein the second time pointis after the first time point; and (c) comparing the levels ofantibodies of the first and second time points, wherein an increase inthe level of antibodies in the later time point compared to the firsttime point indicates that there is a relapse of membranous nephropathy.The increase is at least 5%, at least 10%, at least 20%, at least 30%,50%, at least 100%, at least 200%, at least 300%, at least 500%, atleast 1000%, or more and including all the percentages between 10-1000%.

In an embodiment, provided herein is a method of treatment of membranousnephropathy in a subject, the method comprising removing an antibodythat is reactive to a PLA2R from a sample in a subject. The antibodiesare removed from the blood by immunoabsorption. The sample is returnedback into the subject after the removal of the antibodies.

In an embodiment, provided herein is a method of treatment of membranousnephropathy in a subject, the method comprising administering aneffective amount of PLA2R or fragments thereof or a vector expressing aPLA2R or fragments thereof.

In one embodiment, provided herein is a composition for the treatment ofidiopathic membranous nephropathy, the composition comprisingadministering a PLA2R or fragments thereof.

In one embodiment, provided herein is a use of an effective amount ofPLA2R or fragments thereof or a nucleic acid molecule capable ofexpressing a PLA2R such as a vector expressing a PLA2R or fragmentsthereof for the of treatment of membranous nephropathy in a subject.

In another embodiment, provided herein is a use of an effective amountof PLA2R or fragments thereof or a vector expressing a PLA2R orfragments thereof in the manufacture of a medicament for treatment ofmembranous nephropathy in a subject.

In one embodiment, the fragments suitable for treatment or adsorptionare fragments comprising the CTLDs or CRDs 4, 5 6 of PLA2R.

In one embodiment, provided herein is an immunoassay comprising:contacting a sample from a subject with a PLA2R or PLA2R fragmentthereof; forming an antibody-protein complex between the antibodypresent in a sample with the PLA2R or PLA2R fragment thereof; washing toremove any unbound antibody; adding a detection antibody that is labeledand is reactive to the antibody from the sample; washing to remove anyunbound labeled detection antibody; and converting the label to adetectable signal, wherein the presence of a detectable signal indicatesthe likelihood of MN in the subject.

In one embodiment, provided herein is an immunoassay comprising:contacting a sample from a subject with a PLA2R or PLA2R fragmentthereof; forming an antibody-protein complex between the antibodypresent in a sample with the PLA2R or PLA2R fragment thereof; measuringa light scattering intensity resulting from the formation of theantibody-protein complex wherein the light scattering intensity of atleast 10% above a control light scattering intensity indicates thelikelihood of MN or relapse of MN in the subject. In one embodiment, thecontrol light scattering intensity is that of PLA2R or PLA2R proteinfragment in the absence of a sample from the subject. In anotherembodiment, the control light scattering intensity is that of PLA2R orPLA2R protein fragment in the presence of a sample from a non-MN healthysubject. In another embodiment, the control light scattering intensityis the average light scattering intensity obtained for a population ofnon-MN healthy subjects. Such subject do not have any clinical featuresof the disease as described herein. The increase is at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, 100% and all the percentagesbetween 10-100%. In one embodiment, the light scattering intensity ismeasured in a nephelometer.

In one embodiment, the MN is idiopathic. In other embodiments, thesubject is a human, and the sample is a blood sample. In anotherembodiment, a kidney biopsy is not performed on the subject.

In one embodiment, the PLA2R is a mammalian PLA2R, a human or pig PLA2Rprotein. In one embodiment, the PLA2R or PLA2R protein fragment thereofis deposited or immobilized on a solid support. In another embodiment, aknown amount of a PLA2R or PLA2R protein fragment is deposited orcoupled to a solid support. In other embodiments, the support can be inthe format of a dipstick, a test strip, a latex bead, a microsphere or amulti-well plate.

In one embodiment, the anti-PLA2R auto-antibodies are of the IgGsubclass: IgG1-4. In one embodiment, the detection antibody is labeledby covalently linking to an enzyme, label with a fluorescent compound ormetal, or label with a chemiluminescent compound. In another embodiment,the detection antibody is specifically reactive to the subject, forexample, if the subject is a human, the detection antibody is specificto human.

In one embodiment, the detectable signal is compared to a set ofdetectable signals from a titration curve derived from immunoassays ofknown amounts of PLA2R or fragments in increasing quantity.

In one embodiment, the immunoassay is performed for a plurality ofsamples from a subject obtained over a period of time. The plurality ofsamples is obtained every two or three months for at least a two yearperiod. The detectable signal or light scattering intensity of eachimmunoassay is compared to the detectable signal or light scatteringintensity of samples obtained from a prior time point, wherein areduction of at least 5% of detection signal or light scatteringintensity indicates effective treatment of MN in the subject. Thedecrease is at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%and all the percentages between 10-100%.

In one embodiment, provided herein is a device for identifying thepresence or the level of antibodies that are reactive to a PLA2R in asample from a subject comprising: at least a PLA2R protein or fragmentsthereof; and at least one solid support wherein the PLA2R protein orfragments thereof is deposited on the support. The PLA2R protein orfragments thereof is deposited on the solid support is immobilized onthe support and the solid support is in the format of a dipstick, a teststrip, a latex bead, a microsphere or a multi-well plate.

In one embodiment, the device further comprises a second labeled PLA2Rprotein or fragments thereof which produce a detectable signal. Inanother embodiment, the device further comprises a detection antibody,wherein the detection antibody is specific for the antibodies that arereactive to a PLA2R in the sample of the subject and the detectionantibody produces a detectable signal.

In one embodiment, the devices described herein perform an immunoassaywherein an antibody-protein complex is formed. In one embodiment, theimmunoassay is a serological immunoassay. In another embodiment, theimmunoassay is a nephrelometric immunoassay

In one embodiment, provided herein is the use of the devices describedherein for facilitating the diagnosis of MN in a subject, wherein adetectable amount of antibodies that are reactive to a PLA2R indicateslikelihood of membranous nephropathy in the subject.

In one embodiment, provided herein are kits comprising devices describedherein. In other embodiments, the kits further comprise a detectionantibody, wherein the detection antibody is specific for the antibodiesthat are reactive to a PLA2R in the sample of the subject and produces adetectable signal; a second labeled PLA2R protein or fragments thereofwhich produces a detectable signal; and/or a nephelometer cuvette.

In one embodiment, provided herein is a system comprising: a measuringmodule measuring auto-antibody information comprising a detectablesignal from an immunoassay indicating the presence or level ofantibodies that are reactive to a PLA2R from a sample obtained form asubject; a storage module configured to store data output from themeasuring module; a comparison module adapted to compare the data storedon the storage module with reference and/or control data, and to providea retrieved content, and an output module for displaying the retrievedcontent for the user, wherein the retrieved content the presence ofdetectable amount of antibodies reactive against PLA2R indicates thatthe subject has MN or has a relapse of MN.

In one embodiment, provided herein is a system to facilitate theprognosis evaluation of membranous nephropathy (MN) in a subject,comprising: a determination module configured to receive and outputauto-antibody information to a PLA2R from a sample obtained from asubject, wherein the auto-antibodies information measures the level ofauto antibodies that are reactive to the PLA2R; a storage moduleconfigured to store output information from the determination module; acomparison module adapted to compare the data stored on the storagemodule with reference data and/or control data, and to provide acomparison content, and an output module for displaying the comparisoncontent for the user, wherein if there is no detectable amount of autoantibodies reactive against PLA2R then the subject is in remission or ifthere is a reduction of at least 10% to a prior reading, then thetreatment for MN is effective in the subject. The reduction is at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, 100% and all thepercentages between 10-100%. In one embodiment, the reference or controldata comprises previous data from the same subject wherein the previousdata had indicated detectable amounts of auto-antibodies, detectable byany conventional ELISA or nephelometric immunoassays described hereinand those known in the art. In one embodiment, the reference or controldata comprises the average value of anti-PLA2R auto-antibodies and oneorder of standard deviation obtain from a population of idiopathic MNpatients. The collected sera from these idiopathic MN individuals aredilutd 1:100 and used in Western blot assays. The intensity of thevisible band is quantified by densitometry and the average value and theone order of standard deviation is computed. Ideally, a population hasabout 25 idiopathic MN individuals, preferably more. The statistics, theaverage value and one order of standard deviation can be uploaded to thecomputer system and data storage media.

In one embodiment, provided herein is a computer readable storage mediumcomprising: a storing data module containing data from a sample obtainedfrom a subject that represents a signal level from an immunoassay forantibodies that are reactive to a PLA2R; a comparison module thatcompares the data stored on the storing data module with a referencedata and/or control data, and to provide a comparison content, and anoutput module displaying the comparison content for the user, whereinthe presence of a detectable amount of antibodies reactive against PLA2Rof at least 10% relative to the reference data and/or control dataindicates that the subject has MN or has a relapse of MN. The detectableamount is at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%,200%, 300% or 1000%, including all the percentages between 10-1000%.

In one embodiment, the control data comprises data from a population ofnon-MN healthy individuals, which is the detection signal obtained usinghuman sera at 1:100 dilution with 1×PBS to immuno-react with 0.5 μg ofnative PLA2R, wherein horse-radish peroxidase anti-human IgG antibody isthe labeled deception antibody and the detection signal ischemiluminescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the sera from patients with idiopathic membranousnephropathy (MN) specifically recognize a 200 kDa glomerular antigen bywestern blot. Panel (top) shows human glomerular proteins blotted withfive different sera from patients with idiopathic MN (lanes MN1-5) orwith five sera from patients with other proteinuric conditions (DN,diabetic nephropathy; FS, focal and segmental glomerulosclerosis).

FIG. 1B shows a graphical representation of the specificity of thereactivity of various sera with the 200 kDa antigen. 57% of sera frompatients with idiopathic MN react with the glomerular antigen, whereasthere are no reactive sera from 8 patients with secondary MN, 19 diseasecontrols (other nephrotic conditions or autoimmune diseases), or 23normal controls.

FIG. 1C shows a graphical representation of the specificity of thereactivity of various sera with the 200 kDa (185 kDa) antigen. 72-82% ofsera from patients with idiopathic MN from different regions react withthe glomerular antigen, whereas there are no reactive sera from 12patients with secondary MN, 25 disease controls (other nephroticconditions or autoimmune diseases), or 32 normal controls.

FIG. 2A shows the 200 kDa antigen in human glomeruli is the M-typephospholipase A2 receptor (PLA2R). Human glomerular proteins andrecombinant PLA2R treated with or without PNGase F and western blottedwith either reactive MN serum or a polyclonal antibody raised againstPLA2R are shown.

FIG. 2B demonstrates that human MN sera can immunoprecipitate (IP)PLA2R. Three reactive and three non-reactive sera from MN patients, aswell as three control sera, were used to IP the target antigen from amixture of human glomerular proteins. The immunoprecipitates were thenwestern blotted with antibodies to PLA2R (top) as well as to total humanIgG (bottom panel).

FIG. 2C shows that the glomerular glycoprotein identified by reactive MNsera is the human PLA2R. Whole human serum was used to immunoprecipitate(IP) glomerular proteins, and the IP's were then electrophoresed andWestern blotted with an antibody specific to the M-type PLA2R. The firstfive lanes show IP's with sera that were known to be positive by WB (asin FIG. 1). The 6th lane represents an IP with serum from a patient withMN that was known to be negative. Lanes 7 and 8 show IP's with serumfrom normal volunteers, and in the final lane, human serum was omittedfrom the IP to rule out non-specific binding of glomerular proteins tothe agarose beads.

FIG. 3A shows the epitope on PLA2R is reduction sensitive and elicits anIgG4 predominant response.

FIGS. 3B and 3C shows that the IgG subclass specificity of theauto-antibobodies reactive to PLA2R.

FIG. 4A shows that only IgG eluted from the MN samples identified thenative and recombinant PLA2R.

FIG. 4B shows that the IgG eluted from the MN3 biopsy sample recognizedonly those bands corresponding to PLA2R

FIG. 5A shows the presence of anti-PLA2R antibody in patient serumcorrelates with disease activity. Serial sera were collected from asingle patient with MN who entered remission. The top graph shows adecline in urinary protein levels (diamonds) and an increase in serumalbumin (circles).

FIG. 5B shows the WB in the top panel shows that reactivity to the 200and 150 kDa native and deglycosylated PLA2R is only present in theinitial sample form the same patient of FIG. 5A. Equal loading is shownby the non-specific detection of a 98 kDa band. Total IgG in the serumsamples is shown in the bottom panel, demonstrating a slight increase inIgG as the patient entered remission from MN.

FIG. 6A shows that sera reactivity to PLA2R corresponds to diseaseactivity in patient A with idiopathic MN. The graph shows the decline inprotein in urine upon treatment and the concomitant disappearance ofanti-PLA2 antibodies after treatment commencement in the Western Blot.

FIG. 6B shows that sera reactivity to PLA2R corresponds to diseaseactivity in patient B with idiopathic MN. The graph shows thecorrelation of decline in protein in urine and the disappearance ofanti-PLA2 antibodies prior to and after treatment commencement in theWestern Blot.

FIG. 7 shows schematic diagrams showing the reverse-sandwich ELISA(RS-ELISA) and indirect ELISA.

FIG. 8A (top view) and 8B (side view) shows the schematic diagrams of atest strip for determining the presence and/or level of auto-antibodiesreactive against PLA2R in a fluid sample.

FIG. 9 shows a schematic diagram showing the interpretation of theresults obtained using the test strip shown in FIG. 8.

FIG. 10 shows a schematic diagram of an ELISA plate assay comprisingstandard PLA2R curves.

FIG. 11 shows a schematic diagram of a modified ELISA plate assayutilizing fixed amounts of standard PLA2R protein.

FIG. 12 is a block diagram showing an exemplary system for MN diagnosis.

FIG. 13 is an exemplary set of instructions on a computer readablestorage medium for use with the systems described herein.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in renal diseases, nephrology and molecular biology can be foundin The Merck Manual of Diagnosis and Therapy, 18th Edition, published byMerck Research Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porteret al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);The ELISA guidebook (Methods in molecular biology 149) by Crowther J. R.(2000); Fundamentals of RIA and Other Ligand Assays by Jeffrey Travis,1979, Scientific Newsletters; Immunology by Werner Luttmann, publishedby Elsevier, 2006. Definitions of common terms in molecular biology arefound in Benjamin Lewin, Genes IX, published by Jones & BartlettPublishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); or Methods in Enzymology: Guide to Molecular Cloning TechniquesVol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987), Current Protocols in Molecular Biology (CPMB) (FredM. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols inProtein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley andSons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S.Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of AnimalCells: A Manual of Basic Technique by R. Ian Freshney, Publisher:Wiley-Liss, 5th edition (2005), Animal Cell Culture Methods (Methods inCell Biology, Vol 57, Jennie P. Mather and David Barnes editors,Academic Press, 1st edition, 1998) which are all incorporated byreference herein in their entireties.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

DEFINITIONS OF TERMS

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is described herein. A fragment of PLA2R is ashortened or truncated PLA2R protein. The polypeptide can haveN-terminus or C-terminus truncations and/or also internal deletions.Examples of fragments are fragments comprising the CTLDs or CRDs 4, 5 6of PLA2R. In one embodiment, fragment includes the external domain ofPLA2R, which is the amino acid residues 1-1392 of the human PLA2R (SEQ.ID. NO. 2). Shorter portions of 1-1392 are considered fragments.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carrierof chemicals and compounds commonly used in the pharmaceutical industry.The term “pharmaceutically acceptable carriers” excludes tissue culturemedium.

As used herein, the term “therapeutically effective amount” refers tothat amount of active agent that can reduce the amount of solubleauto-antibodies available for binding to PLA2R.

As used herein, the term “treat’ or treatment” refers to reducing oralleviating at least one adverse effect or symptom associated withmedical conditions that are associated with membranous nephropathy.These include reducing the amount of auto-antibodies against PLA2Rprotein, reducing, inhibiting or stopping the production ofauto-antibodies against PLA2R, suppression of the immune system, andreducing the inflammation and degradation/damage associated with theactivities of the auto-antibodies when they are bound to the kidneyglomeruli.

The term “subject” as used herein includes, without limitation, a human,mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee,baboon, or rhesus. In one embodiment, the subject is a mammal. Inanother embodiment, the subject is a human.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

As used herein, the term “idiopathic MN” is currently used to describeMN that is not caused by any known secondary etiology such as hepatitisB or lupus prior to the present discovery. On the basis of the presentdiscovery, “idiopathic MN” refers to PLA2R-associated MN or any otherfuture designation for what is now called idiopathic MN and isassociated with anti-PLA2R antibodies.

The term C-type lectin (“CTLD”) or carbohydrate-recognition domain(“CRD”) with respect to the domains in PLA2R are used interchangeably.

Embodiments of the invention are based on the discovery that sera ofpatients with MN contain antibodies that are reactive against the M-typePLA2R that is found in the glomerulus. Idiopathic membranous nephropathy(MN) is considered to be an autoimmune disease targeting the glomerulus,yet the major target antigen has remained elusive. The inventorsscreened sera from patients with MN for reactivity against humanglomerular proteins by western blot (WB) and found a commonly-detected200 kDa glycoprotein. The inventors then proceeded through partialpurification and mass spectrometric analyses to identify this 200 kDaglycoprotein. It is the M-type PLA2R. Soluble and membrane boundisoforms of PLA2R1 (180 kDa) are found. In vivo, PLA2R is expressed inkidneys. This native PLA2R from the glomeruli extract has been furthercharacterized and now is determined to be approximately 185 kDa on aprotein gel. Upon deglycosylation in the method described herein, it isapproximately 145 kDa.

The inventors found that about 70-82% of patients with MN had antibodiesthat are reactive with a recombinant PLA2R (rPLA2R) by WB, and the humansera are able to immunoprecipitate (IP) the native protein from extractsof normal human glomeruli. Control sera from normal volunteers andnephrotic controls, as well as sera from MN patients previouslynon-reactive by WB, do not identify rPLA2R or IP the native protein. Themajority of the reactive immunoglobulin in patients' sera is IgG4, thesubclass that predominates in glomerular deposits in idiopathic MN. Theinventors show that PLA2R is present in podocytes, as detected byimmunofluorescence on cryosections of human kidney. Moreover, both PLA2Rand IgG4 co-localize on biopsy specimens from patients with MN in a finegranular pattern typical of the subepithelial deposits characteristic ofthe disease. While not wishing to be bound by theory, theauto-antibodies in the patients' sera against PLA2R bind to the PLA2R inthe kidney glomerulus, causing structural damage to the kidneys andimpair kidney function. While not wishing to be bound by theory, thebinding of the auto-antibodies to PLA2R cause sublethal injury to thepodocytes and induces massive proteinuria. With the major target ofidiopathic MN antibodies now being identified as PLA2R, this would allowfor earlier and less invasive detection of the disease with animmunoassay designed to measure circulating autoantibodies, and alsolead to a means for monitoring response to treatment. In addition, PLA2Rauto-antibodies of the IgG1, IgG2, and IgG3 subclasses were alsodetected. The inventors also found that the auto-antibodies are reactiveagainst mammalian PLA2R, such as the human, rabbit, and pig PLA2R.

The nephrotic syndrome, characterized by edema and large amounts ofprotein in the urine, is a relatively common disorder of the kidney thathas many potential causes, including membranous nephropathy (MN), focaland segmental glomerulosclerosis, minimal change disease, diabeticnephropathy, membranoproliferative glomerulonephritis, as well as othercauses. Although there are non-specific treatments that ameliorate someof the signs and symptoms of the nephrotic condition, specific knowledgeof the underlying disease is usually necessary to guide definitivetreatment. When a patient with the nephrotic syndrome is initiallyevaluated as an outpatient or in the hospital, a panel of blood tests isusually ordered by the physician to look for potential causes that aredetectable by serology (for example, the presence of anti-nuclearantibodies (ANA) and/or anti-double stranded DNA antibodies in thecontext of typical findings in the urinary sediment would suggest lupusnephritis). There is currently no serologic test to identify MN, anddiagnosis relies exclusively on kidney biopsy, an invasive procedurerequiring overnight hospitalization in many institutions and one thatcan be complicated by major internal bleeding. MN can be caused by anumber of secondary factors, such as systemic lupus erythematosus,hepatitis B, or syphilis, and blood tests are routinely sent to look forthese causes (ANA, anti-hepatitis B antigens, rapid plasma reagin (RPR),respectively). However, in the United States, MN is more often primary,or idiopathic, in origin and as mentioned above, no blood test for thisform currently exists, since the antigen targeted in this autoimmunedisease had not been identified until this point.

Membranous nephropathy, a frequent cause of adult nephrotic syndrome, iswidely felt to be an autoimmune disease despite ignorance of thelong-sought target antigen. Much of the support for an autoimmune basisfor MN comes from decades of research on the rat model of Heymannnephritis (FIN), which is virtually identical at the pathological levelto the human disease. In HN, the target antigen is megalin, a moleculein the LDL receptor family that is responsible for semi-selective uptakeof proteins in the kidney. It is present, in rats but not in humans, onthe podocyte, and circulating antibodies have been shown to bind theprotein in situ, leading to shedding of the antibody-antigen complexesinto the glomerular basement membrane, leading to the subepithelialdeposits characteristic of both HN and MN.

Since the sera from control healthy individuals and non-MN nephropathypatients do not contain or have very very low amount or undetectableamount of auto-antibodies that react with PLA2R unlike the sera of MNpatients, the detection of the presence of PLA2R antibodies can be usedas a diagnostic tool for MN. A simple blood sample can be used to testfor and detect antibodies reactive against PLA2R. Such a method would behighly favorable over the current diagnostic method of a kidney tissuebiopsy which is an invasive technique. Accordingly, in one embodiment,provided herein is a method of diagnosing membranous nephropathy in asubject, the method comprising detecting the presence of antibodies thatare reactive to a phospholipase A2 receptor, wherein the antibodies arefound in a sample from a subject. The antibodies can be detected by animmunoassay wherein an antibody-protein complex is formed. Theimmunoassay can be a serological immunoassay or a nephelometricimmunoassay. The subject is a mammal, such as a dog, a cat, or a human.Healthy subjects who do not have MN or do not have any symptoms relatedto MN, e.g. protein in the urine, have undetectable auto-antibodies tophospholipase A2 receptor (PLA2R). When antibodies that are reactive toPLA2R are detected in a subject suspected of having MN, e.g. havingprotein in the urine, the presence of the anti-PLA2R antibodiesindicates the likelihood of the subject having MN. As an examplary, byundetectable amount of anti-PLA2R auto-antibodies, it means that novisible band is observed in the Western Blot analysis performed asdescribed in Example 1, wherein human serum is diluted 1:100 and used inblot assays described herein. By very very low amount ofanti-PLA2Rauto-antibodies, the low amount is the average amount found ina population of non-MN healthy subjects. The terms “subjects”,“individuals” or “patients” are used interchangeably. The amount ofanti-PLA2R auto-antibodies in a healthy non-MN individual or apopulation of healthy non-MN individuals as determined by conventionalELISA or Western blot set forth in Example 1 can be considered as thebackground, reference or the control level. The collected sera from thehealthy non-MN individuals are diluted 1:100 and used in Western blotassays. The intensity of the visible band is quantified by densitometryand the average value and the one order of standard deviation iscomputed. Ideally, a population has about 25 healthy non-MN individuals,preferably more. The statistics, the average value and one order ofstandard deviation can be uploaded to the computer system and datastorage media. Patients having at least 10% more than this averageamount of anti-PLA2R auto-antibodies is likely to have MN, especially ifthe patient is also presents the clinical features of the disease, e.g.proteinuria and nephrotic syndrome.

In one embodiment, provided herein is an immunoassay comprising:contacting a sample from a subject with a PLA2R or PLA2R fragmentthereof; forming an antibody-protein complex between the antibodypresent in a sample with the PLA2R or PLA2R fragment thereof; washing toremove any unbound antibody; adding a detection antibody that is labeledand is reactive to the antibody from the sample; washing to remove anyunbound labeled detection antibody; and converting the label to adetectable signal, wherein the presence of a detectable signal indicatesthe likelihood of MN in the subject.

In some embodiments, the detection antibody is labeled by covalentlylinking to an enzyme, label with a fluorescent compound or metal, labelwith a chemiluminescent compound. For example, the detection antibodycan be labeled with catalase and the conversion uses a colorimetricsubstrate composition comprises potassium iodide, hydrogen peroxide andsodium thiosulphate; the enzyme can be alcohol dehydrogenase and theconversion uses a colorimetric substrate composition comprises analcohol, a pH indicator and a pH buffer, wherein the pH indicator isneutral red and the pH buffer is glycine-sodium hydroxide; the enzymecan also be hypoxanthine oxidase and the conversion uses a colorimetricsubstrate composition comprises xanthine, a tetrazolium salt and4,5-dihydroxy-1,3-benzene disulphonic acid.

In one embodiment, the detection antibody is specifically reactive onlyto the specie of the subject. For example, if the human, then thedetection antibody is an anti-human antibody. If the subject is a horse,then the detection antibody is an anti-horse antibody. If the subject isa dog, then the detection antibody is an anti-dog antibody.

In one embodiment, the detectable signal is compared to a set ofdetectable signals from a titration curve derived from immunoassays ofknown amounts of PLA2R or fragments in increasing quantity.

In another embodiment, provided herein is an immunoassay comprising:contacting a sample from a subject with a PLA2R or PLA2R fragmentthereof; forming an antibody-protein complex between the antibodypresent in a sample with the PLA2R or PLA2R fragment thereof; measuringa light scattering intensity resulting from the formation of theantibody-protein complex wherein the light scattering intensity of atleast 10% above a control light scattering intensity indicates thelikelihood of MN or relapse of MN in the subject. The control lightscattering intensity is that of PLA2R or PLA2R protein fragment in theabsence of sample. In another embodiment, the control light scatteringintensity is that of PLA2R or PLA2R protein fragment in the presence ofa sample from a non-MN healthy subject. In another embodiment, thecontrol light scattering intensity is the average light scatteringintensity obtained for a population of non-MN healthy subjects. In oneembodiment, the light scattering intensity is measured in anephelometer. The increase is at least 20% at least 30%, at least 50%,at least 100%, at least 200%, at least 300%, at least 500%, at least1000%, or more and including all the percentages between 10-1000%.

In one embodiment, the MN in the subject is idiopathic and a kidneybiopsy is not performed on the subject. In one embodiment, the subjectis a human and the sample from the subject is a blood sample, e.g. serumor plasma.

In one embodiment, the PLA2R is a mammalian PLA2R, such as human or pigPLA2R.

In one embodiment, the anti-PLA2R antibodies are of the IgG subclass:IgG1-4.

In one embodiment, the immunoassay is a serological immunoassay.

In some embodiments, the PLA2R or PLA2R protein fragment thereof isdeposited on a solid support or it can be coupled to immobilize theprotein on the support. The support can be is in the format of adipstick, a test strip, a latex bead, a microsphere or a multi-wellplate. In one embodiment, a known amount of a PLA2R or PLA2R proteinfragment is deposited or coupled to a solid support. The range ofprotein is between 0.1 ng-1 mg.

In one embodiment, the immunoassay described herein is performed for aplurality of samples from a subject obtained over a period of time. Inone embodiment, the pluralities of samples are obtained every two orthree months for at least a two year period. For example, in a bloodsample is collected from a patient diagnosed with MN every three monthsto monitor the progress of the condition and the effectiveness of theimmunosuppressive treatment. The result of immunoassay of each bloodsample is recorded and the date of sample noted. The result ofimmunoassay of each blood sample is compared to that obtained for aprevious blood sample taken three months earlier. It can also becompared to the results obtained during initial diagnosis before thestart of immunosuppressive treatment.

In one embodiment, the detectable signal or light scattering intensityof each immunoassay is compared to the detectable signal or lightscattering intensity of a sample obtained from a prior time point,wherein a reduction of at least 5%, at least 10% or more of detectablesignal or light scattering intensity indicates effective treatment of MNin the subject, including a reduction of at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, 100% and all the percentages between 10-100%. The prior timepoint can be the immediate consecutive prior time point or an earliertime point, e.g. six months earlier or that obtained during initialdiagnosis before the start of immunosuppressive treatment.

In one embodiment, provided herein is a method of prognosis evaluationin a subject being treated for membranous nephropathy, the methodcomprising: (a) determining at a first time point in a sample from asubject a level of antibodies that are reactive to a PLA2R; (b)determining at a second time point in a sample from the same subject alevel of antibodies that are reactive to a PLA2R, wherein the secondtime point is after the first time point; and (c) comparing the levelsof antibodies obtained for the two time points, wherein a decrease inthe level of antibodies in the second time point compared to the firsttime point indicates that the treatment is effective. The level of theantibodies can be detected by an immunoassay wherein an antibody-proteincomplex is formed. The subject has initially been diagnosed with MN andhas a detectable amount of auto-antibodies against PLA2R. Upontreatment, for example, with immunosuppressive therapy, over time, thereis a decrease in the amount of detectable auto-antibodies against PLA2R.In an ideal case, the amount of auto-antibodies should fall below thedetectable level of the detection methods described herein and thesubject is deemed to be in remission for the disorder. A decrease in thelevel of antibodies in the second time point compared to the first timepoint is at least 5%, at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, 100% and all the percentages between 10-100% drop in the titer ofauto-antibodies against PLA2R in the serum of second time point comparedto the first time point. Below the detection limit is when the level ofantibodies is reduced to between 95-100% or more compared to the firsttime point when the subject was initially diagnosed with MN and notreatment has be implemented. The assay used is identical for all thesamples collected different time points from the subject. Decreasingtiter of auto antibodies indicate that the treatment is effective in thesubject.

In other embodiments, there is no decrease in the level of antibodies inthe second time point compared to the first time point. Instead, therecan be an increase or a stable level of antibodies.

In one embodiment, there is an increase in antibody level in the secondtime point compared to the first time point and the first time point hasno detectable auto-antibodies. This indicates that the patient hasrelapsed and the MN has recurred.

In one embodiment, there is an increase in antibody level in the secondtime point compared to the first time point and the first time point hasdetectable auto-antibodies. This indicates worsening of the diseaseand/or lack of efficacious treatment. The increase is at least 30%, atleast 50% at least 100%, at least 200%, at least 300%, at least 500%, atleast 1000%, or more and including all the percentages between 10-1000%.

In one embodiment, the stable level of antibody, wherein theauto-antibodies detectable at the second and first time points arecomparably similar within statistical analysis variances, about 1%, 2%,3%, 4%, 5% and all the percentages between 1%-5% deviation from thelevel of auto-antibodies from the first time point. The stable level ofantibody indicates stable disease, wherein the treatment has been ofinsufficient duration (i.e., that it should be continued if clinicallyindicated) or is ineffective.

As used herein, the term “auto-antibodies” and “antibodies” againstPLA2R are used interchangeably.

In other embodiments, the immunoassays comprise beads coated with nativeor recombinant PLA2R protein as described in Binder S R., Lupus. 2006,15:412-21. Commonly used are polystyrene beads that are dyed toestablish a unique identity. Detection is performed by flow cytometry.Autoantibody detection using multiplex technologies. Other types ofbead-based immunoassays are well known in the art, e.g. laser beadimmunoassays and related magnetic bead assays (Fritzler, Marvin J;Fritzler, Mark L, Expert Opinion on Medical Diagnostics, 2009, pp. 3:81-89).

In another embodiment, provided herein is a method of prognosisevaluation in a subject for membranous nephropathy, the methodcomprising: (a) determining at a first time point in a sample from asubject a level of antibodies that are reactive to a PLA2R; and (b)determining at a subsequent time point, i.e. at a second time point in asample from the same subject a level of antibodies that is reactive to aPLA2R, wherein the second time point is after the first time point;wherein no detectable auto-antibodies against PLA2R at the second timepoint compared to the first time point indicates that the subject is inremission for MN. The level of the antibodies can be detected by animmunoassay wherein an antibody-protein complex is formed. The detectionlimit is when the level of antibodies is reduced to between 95-100% andbeyond more compared to that of the first time point.

In a further embodiment, provided herein is a method of prognosisevaluation in a subject for membranous nephropathy, the methodcomprising: (a) determining at a first time point in a sample from asubject a level of antibodies that are reactive to a PLA2R; (b)determining at a subsequent time point, i.e. at a second time point in asample from the same subject a level of antibodies that is reactive to aPLA2R, wherein the second time point is after the first time point; and(c) comparing the levels of antibodies of the two time points, whereinan increase of at least 5% in the level of antibodies at the second timepoint compared to the first time point indicates that there is relapseof MN. The level of the antibodies can be detected by an immunoassaywherein an antibody-protein complex is formed. The increase is at least10%, at least 20%, at least 30%, at least 50%, at least 100%, at least200%, at least 300%, at least 500%, at least 1000%, or more andincluding all the percentages between 10-1000%.

In one embodiment, the subject has been successfully been treated forMN, has no detectable auto-antibodies against PLA2R in blood circulationand is currently not on under any treatment for MN. In this subject, thefirst time point has no detectable auto-antibodies. The subject hadpreviously been diagnosed with MN and has a detectable amount ofauto-antibodies against PLA2R. Upon treatment, for example, withimmunosuppressive therapy, over time, the amount of auto-antibodiesagainst PLA2R drops to below the detectable level of the detectionmethods described herein and the subject is in remission for MN. There-emergence of a detectable amount of auto-antibodies against PLA2R,and the gradual increase of the auto-antibodies over time indicates thatMN has recurred in the subject.

In another embodiment, the subject is currently being treated for MN andhas detectable auto-antibodies against PLA2R in blood circulation. Anincrease in the level of antibodies at the second time point compared tothe first time point indicates that the disease condition isdeteriorating and the treatment at the current regime is not effectivein slowing/stopping the disease.

In another embodiment, the subject is currently being treated for MN,has detectable auto-antibodies against PLA2R in blood circulation andthe level of auto-antibodies in the first and second time points arecomparably similar within statistical analysis variances. This indicatethat the subject has a steady level of auto-antibodies during treatment,indicating that the treatment has been of insufficient duration (i.e.,that it should be continued if clinically indicated) or is ineffective.

In one embodiment, the MN is idiopathic. The subject who is beingsuspected of having MN has tested negative for the usual causes of MN,for example, systemic lupus erythematosus, hepatitis B, and syphilis. Askilled clinician would have, by the process of elimination, ruled outall possible causes for MN. What is left is the tentative diagnosis ofMN from an obscure or unknown cause, such as possibly auto-immuneauto-antibodies against PLA2R. The non-invasive diagnostic methoddescribed herein can then be applied to such a subject for confirmation.

In one embodiment, the method of diagnosing MN described herein isapplied to a patient who presents symptoms of MN without havingundergone the routine screening to rule out all possible causes for MN.The methods described herein can be part of the routine set of testsperformed on a patient who presents symptoms of MN such as proteinuriafor diagnostic purposes. The test can be an immunoassay wherein anantibody-protein complex is formed, e.g. serological immunologicalassays. Such patients have not been biopsied for the confirmatorydiagnosis of MN. Similarly, methods described herein can be part of theroutine set of serological immunological tests performed for a patientwho already is known to have MN by biopsy or by serological testing, isbeing treated for MN. The methods are useful for the monitoring ofimmunosuppressive therapies efficacy and prognostic evaluation in thepatient.

In one embodiment, the subject is a mammal. In another embodiment, thesubject is a human. It is envisioned that the methods described hereinare applicable to any mammal that has kidneys, expresses PLA2R and hasan immune system that comprises antibodies.

In one embodiment, the subject who is being suspected of having MN hasnot undergone a kidney biopsy for a confirmatory diagnosis of MN.

The inventors have found that the antibodies in the sera of MN patientswere immunologically reactive against mammalian PLA2R such as the human,rabbit and PLA2R. Therefore, encompassed herein, the methods describedherein comprise detecting antibodies that are reactive against the humanphospholipase A2 receptor or pig PLA2R. As used herein, the term“reactive against”, “react to” or “reactive with” refers to theantibodies recognizing the human or pig PLA2R and binding to the PLA2R.The recognition and binding are the standard antibody-antigeninteractions that are well characterized by biochemistry and immunology.

In one embodiment, the sample from the subject is a blood sample. Inother embodiments, the sample is whole blood, serum, or plasma.

In one embodiment, the antibodies are of the IgG4 subclass. In otherembodiments, the PLA2R auto-antibodies are of the subclass IgG3 andIgG1. In yet other embodiments, PLA2R auto-antibodies are of the IgGsubclasses: IgG1-4.

In one embodiment, the treatment for MN is an immunosuppressive therapy,for example, cyclosporin, tacrolimus, azathioprine, infliximab,omalizumab, daclizumab, adalimumab, eculizumab, efalizumab, natalizumab,omalizumab and rapamycin. In a further embodiment, the immunosuppressivetreatment for MN additionally includes but is not limited tocyclophosphamide, chlorambucil, and rituximab.

In some embodiments, for the methods described herein, the detection ofauto-antibodies against PLA2R is performed by a serological immunoassaysuch as an enzyme-linked immunosorbent assays (ELISAs). ELISAs and otherimmunoassays known in the art are generally created using standardprotocols, with the major variation being the target, or capture,antigen. For the methods described herein, the antigen is human PLA2R,pig PLA2R, or fragments thereof. Recombinant full-length PLA2R expressedin a mammalian or insect cell line can be purified and used as thecapture antigen. The protein can be expressed with an N- or C-terminalFLAG tag to facilitate purification from other cell-line derivedproteins. ELISA plates will be coated with PLA2R or a fragment at aconstant concentration. The plate can be blocked with bovine casein orserum albumin or other blocking agents to prevent nonspecific binding ofthe samples. Human serum to be tested can be added to the wells atstandard dilutions (1:40, 1:80, 1:160, etc.) followed by routine washes.Bound IgG can be detected with a secondary anti-human IgG antibodylinked to horseradish peroxidase. After a series of washes, colorimetricsubstrate can be added to all wells and developed. The ELISA plate canbe read on a micro titer plate reader. Using MN serum samples that areknown to be positive or negative, as well as serum from normal humanvolunteers, it is possible to establish appropriate cut-off titers todefine what will constitute a positive test result. Healthy subjects whodo not have MN or do not have any symptoms related to MN, e.g. proteinin the urine, have undetectable auto-antibodies to PLA2R. Whenantibodies that are reactive to PLA2R are detected in a subjectsuspected of having MN, e.g. having protein in the urine, the presenceof the anti-PLA2R antibodies indicates the likelihood of the subjecthaving MN.

In one embodiment, an ELISA can provide a simple serological assay forimmunologically-active MN, i.e. active MN with auto-antibodies againstPLA2R. The simple serological assay can be ordered with other widelyused diagnostic or otherwise informative blood tests in patients withheavy proteinuria, such as anti-nuclear antibodies, anti-hepatitis-B and-C antibodies, and complement C3 and C4 levels. In the cases of patientswho test positive in this assay, biopsy of the kidney may not benecessary to guide treatment, unless other atypical features are presentthat might warrant a biopsy. In those patients who test negative andstill have unexplained proteinuria, however, a renal biopsy is stillindicated. An immunoassay as described can be considerably cheaper thanrenal biopsy, which often requires an overnight admission to thehospital. It is also much more convenient for both the patient andphysician.

It is a known fact that proteinuria can persist (transiently orpermanently) in MN patients even after the immunological disease isover, due to structural changes in the glomerulus. Reliance on levels ofprotein excretion alone can lead to the treatment of patients with toxicimmunosuppressive drugs much longer than necessary. Thus, monitoring fordisappearance of auto antibodies with the described ELISA will helpdefine a transition point in the treatment of membranous nephropathywhen immunosuppressive therapy should be stopped but anti-proteinurictherapy (e.g., angiotensin-converting enzyme inhibitors) should becontinued.

In one embodiment, provided herein is a method of treatment ofmembranous nephropathy in a subject, the method comprising administeringan effective amount of PLA2R or fragments thereof or a vector expressinga PLA2R or fragments thereof. By providing soluble PLA2R or fragmentsthereof, the soluble protein can function as decoy antigens andsequester the auto-antibodies away from the PLA2R in the renalglomeruli, thereby reducing the potential damage to the kidney. ThePLA2R can be the human or pig PLA2R. In one embodiment, the fragmentssuitable for treatment or adsorption of the auto-antibodies to PLA2Rfrom the serum are fragments comprising the CTLDs or CRDs 4, 5 6 ofPLA2R. In another embodiment, the fragments comprise the extracellulardomain of human or pig PLA2R. In one embodiment, the fragment is SEQ.ID. NO. 5 or smaller portions of SEQ. ID. NO. 5, such as at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95 including all thepercentages between 10-95%. Also comtempted are peptides between 10-50amino acid residues derived on the sequence of SEQ. ID. NO. 5 that canbe used in the treatment of MN. In one embodiment, a cocktail of severalpeptides is used for treatment. Envisioned peptides can be fused withother proteins for longer serum half-life, tandemly linked peptides orcircular peptides.

In one embodiment, the membranous nephropathy is idiopathic. The subjecthas tested positive for antibodies reactive against a PLA2R. In oneembodiment, the auto-antibodies are reactive to the human PLA2R or pigPLA2R.

In another embodiment, provided herein is a method of treatment ofmembranous nephropathy in a subject, the method comprising removing anantibody that is reactive to a PLA2R from a sample in a subject. Theantibodies are removed from the blood by immunoabsorption with PLA2R orfragments thereof as the antigen, and the sample is returned back intothe subject after the removal of the antibodies. The fragments suitableadsorption are fragments comprising the CTLDs or CRDs 4, 5 6 of PLA2R.For the human PLA2R receptor 1 isoform 1 precursor (GENBANK™ AccessionNo. NP_(—)031392.3, SEQ. ID. NO. 2), a suitable fragment can be aminoacid residues 650 to 1100 which consist of the CRDs 4, 5 6 of PLA2R.

In one embodiment, provided herein is a use of an effective amount ofPLA2R or fragments thereof or a vector expressing a PLA2R or fragmentsthereof for the of treatment of membranous nephropathy in a subject.

In one embodiment, provided herein is a use of an effective amount ofPLA2R or fragments thereof or a vector expressing a PLA2R or fragmentsthereof in the manufacture of a medicament for treatment of membranousnephropathy in a subject.

In one embodiment, the sample is blood. In other embodiments, the sampleis serum or plasma. In one embodiment, the subject is a human and themembranous nephropathy is idiopathic. The auto-antibodies are reactiveto the human PLA2R or the pig PLA2R receptor.

In one embodiment, the antibodies are of the IgG4 subclass. In otherembodiments, the PLA2R auto-antibodies are of the subclass IgG3, IgG2,and IgG1. In yet other embodiments, PLA2R auto-antibodies are of the IgGsubclasses: IgG1-4. The immunoabsorption of auto-antibodies againstPLA2R helps reduce the amount of circulating auto-antibodies and therebyreducing the potential damage to the kidney. This treatment can beapplied initially after immunological confirmation of the presence ofthe auto-antibodies reactive against PLA2R and before the start of anyimmunosuppressive therapy. This is especially useful during this earlyperiod before the immunosuppressive therapy can have an effect on theimmune system and production of auto-antibody in the subject.

In one embodiment, immunoabsorption of auto-antibodies against PLA2R canoccur by passing the blood, serum or plasma over immobilized PLA2R.Recombinant human or pig PLA2R or fragments can be immobilized on inertand sterile matrices that are known in the art, such as sepharose. Theauto-antibodies against PLA2R will bind to the immobilized PLA2R orfragments and remind bound to the matrix indirectly. The blood, serum orplasma is then collected. This resultant blood, serum or plasma shouldhave no detectable or reduced auto-antibodies against PLA2R. Theimmunoabsorption procedure should be conducted under sterile conditions.The collect blood, serum or plasma that is now depleted ofauto-antibodies against PLA2R can now be transfused back into thepatient.

Devices, Kits, Computer System and Computer Data Storage

In one embodiment, provided herein are devices for identifying thepresence or the level of antibodies that are reactive to a PLA2R in asample from a subject comprising: at least a PLA2R protein or fragmentsthereof; and at least one solid support wherein the PLA2R protein orfragments thereof is deposited on the support. In one embodiment, thePLA2R protein or fragments thereof that is deposited on the solidsupport is immobilized on the support. In one embodiment, the PLA2Rprotein is a human or pig PLA2R protein. In one embodiment, the solidsupport is in the format of a dipstick, a test strip, a latex bead, amicrosphere or a multi-well plate.

In one embodiment, the subject is a human and a kidney biopsy is notperformed in the subject. In one embodiment, the sample from the subjectis a blood sample.

In other embodiments, the devices or kits described herein can furthercomprise a second labeled PLA2R protein or a fragment thereof whichproduces a detectable signal; a detection antibody, wherein thedetection antibody is specific for the antibodies that are reactive to aPLA2R in the sample of the subject and the detection antibody produces adetectable signal; or a nephelometer cuvette.

In one embodiment, the device performs an immunoassay wherein anantibody-protein complex is formed, such as a serological immunoassay ora nephelometric immunoassay

In some aspects, the devices described herein facilitate the diagnosisof membranous nephropathy in a subject, wherein a detectable amount ofantibodies that are reactive to a PLA2R indicates likelihood ofmembranous nephropathy in the subject.

In one embodiment, provided herein are kits that comprise devicesdescribed herein and a detection antibody, wherein the detectionantibody is specific for the antibodies that are reactive to a PLA2R inthe sample of the subject and produces a detectable signal. In oneembodiment, the kit can include a second labeled PLA2R protein or afragment thereof which produces a detectable signal. In furtherembodiments, the kit includes a nephelometer cuvette.

Any solid support can be used, including but not limited to,nitrocellulose membrane, nylon membrane, solid organic polymers, such aspolystyrene, or laminated dipsticks such as described in U.S. Pat. No.5,550,375. The use of “dip sticks” or test strips and other solidsupports have been described in the art in the context of an immunoassayfor a number of antigens. Three U.S. patents (U.S. Pat. No. 4,444,880,issued to H. Tom; U.S. Pat. No. 4,305,924, issued to R. N. Piasio; andU.S. Pat. No. 4,135,884, issued to J. T. Shen) describe the use of “dipstick” technology to detect soluble antigens via immunochemical assays.The apparatuses and methods of these three patents broadly describe afirst component fixed to a solid surface on a “dip stick” which isexposed to a solution containing a soluble antigen that binds to thecomponent fixed upon the “dip stick,” prior to detection of thecomponent-antigen complex upon the stick. The “dip stick” technology canbe easily adapted for the present invention by one skilled in the art.In the invention described herein, the antigen PLA2R is deposited on thesupport and the auto-antibody is to be detected.

Examples of kits include but are not limited to ELISA assay kits, andkits comprising test strips and dipsticks. In an ELISA kit, an excessamount of PLA2R antigen, in, is immobilized on a solid support. A samplecontaining an unknown amount of auto-antibodies to PLA2R is added to theimmobilized PLA2R, resulting in the formation of a complex consisting ofthe protein and the antibody. The complex is detected by a labeledsecond antibody that is also specific for the auto-antibody. The amountof label detected is a measure of the amount of auto antibody present inthe sample (see example 3).

In some embodiments of the kits described herein, the kit comprises atest strip or a dipstick.

In some embodiments of the kits described herein, the labeled antibodiesare detectably labeled by enzyme labeling, fluorescent labeling, biotinlabeling or radioisotope labeling. Other labels include but are notlimited to colloidal gold and latex beads. The latex beads can also becolored. Method of labeling antibodies are known in the art, forexample, as described in “Colloidal Gold. Principles. Methods andApplications”, Hayat M A (ed.) (1989-91). Vols 1-3, Academic press,London; in “Techniques in Immunocytochemistry”, Bullock G R and PetruszP (eds) (1982-90) Vols 1, 2, 3, and 4, Academic Press, London; in“Principles of Biological Microtechnique”, Baker J R (1970), Methuen,London; Lillie R D (1965), Histopathologic Technique and practicalHistochemistry, 3rd ed, McGraw Hill, New York; Berryman M A, et al(1992), J. Histochem Cytochem 40, 6, 845-857, all of which areincorporated hereby reference in their entirety.

In a typical colloidal gold labeling technique, the unique red color ofthe accumulated gold label, when observed by lateral or transverse flowalong a membrane on which an antigen is captured by an immobilizedantibody, or by observation of the red color intensity in solution,provides an extremely sensitive method for detecting sub nanogramquantities of proteins in solution. A colloidal gold conjugate consistsof a suspension of gold particles coated with a selected protein ormacromolecule (such as an antibody or antibody-based moiety). The goldparticles may be manufactured to any chosen size from 1-250 nm. Thisgold probe detection system, when incubated with a specific target, suchas in a tissue section, will reveal the target through the visibility ofthe gold particles themselves. For detection by eye, gold particles willalso reveal immobilized antigen on a solid phase such as a blottingmembrane through the accumulated red color of the gold sol. Silverenhancement of this gold precipitate also gives further sensitivity ofdetection. Suppliers of colloidal gold reagents for labeling areavailable from SPI-MARK™. Polystyrene latex Bead size 200 nm coloredlatex bead coated with antibody SIGMA ALDRICH®, Molecular Probes, BangsLaboratory Inc., and AGILENT® Technologies.

In other embodiments of the kits described herein, at least one of thelabeled antibodies comprises an enzyme-labeled antibody. The anti-PLA2Rthat is bound and captured by the immobilized PLA2R on the solid support(e.g. microtiter plate wells) is identified by adding a chromogenicsubstrate for the enzyme conjugated to the anti-antibody, e.g.anti-human IgG, and color production detected by an optical device suchas an ELISA plate reader.

Other detection systems can also be used, for example, abiotin-streptavidin system. Quantification is determined using astreptavidin-peroxidase conjugate and a chromagenic substrate. Suchstreptavidin peroxidase detection kits are commercially available, e.g.from DAKO; Carpinteria, Calif.

Detection antibodies and PLA2R can alternatively be labeled with any ofa number of fluorescent compounds such as fluorescein isothiocyanate,europium, lucifer yellow, rhodamine B isothiocyanate (Wood, P. In:Principles and Practice of Immunoassay, Stockton Press, New York, pages365-392 (1991)) for use in immunoassays. In conjunction with the knowntechniques for separation of antibody-antigen complexes, thesefluorophores can be used to quantify the level of auto antibodies. Thesame applies to chemiluminescent immunoassay in which case antibody orPLA2R can be labeled with isoluminol or acridinium esters (Krodel, E. etal., In: Bioluminescence and Chemiluminescence: Current Status, JohnWiley and Sons Inc. New York, pp 107-110 (1991); Weeks, I. et al., Clin.Chem., 29:1480-1483 (1983)). Radioimmunoassay (Kashyap, M. L. et al., J.Clin. Invest., 60:171-180 (1977)) is another technique in whichdetection antibody can be used after labeling with a radioactive isotopesuch as ¹²⁵I. Some of these immunoassays can be easily automated by theuse of appropriate instruments such as the IMX™ (Abbott, Irving, Tex.)for a fluorescent immunoassay and Ciba Coming ACS 180™ (Ciba Corning,Medfield, Mass.) for a chemiluminescent immunoassay.

In some embodiments, the kits described herein further comprisestandards of known amounts of the PLA2R or fragments thereof.

In some embodiments, the kits described herein further comprisereference values of the levels of anti-PLA2R antibodies. The referencevalues are average levels of anti-PLA2R antibodies in samples from apopulation of non-MN healthy humans. Reference values can be provided asnumerical values, or as standards of known amounts or titer ofanti-PLA2R antibodies presented in pg/ml-μg/ml.

In some embodiments, the kits described herein further comprise at leastone sample collection container for sample collection. Collectiondevices and container include but are not limited to syringes, lancets,BD VACUTAINER® blood collection tubes.

In some embodiments, the kits described herein further compriseinstructions for using the kit and interpretation of results. Forexample, a chart showing FIG. 9 interpretation of results.

As an exemplary, a typical ELISA-based kit assay would involveddispensing a sample containing the serum into microtiter plate wells,preferably in duplicates or triplicates (as in FIG. 11). The wells arecoated with immobilized PLA2R. In addition, a fixed amount of thestandard anti-IgG provided with the kit is also dispensed into referencewells in the microtiter plate, also preferably in duplicates ortriplicates, according the kit's instruction. That fixed amount of thestandard anti-IgG corresponding to at least two fold of the referencevalue of the anti-PLA2R auto-antibodies normally present in healthysubjects. A second fixed amount of the standard anti-IgG correspondingto two fold lower than of the reference value of the anti-PLA2Rauto-antibodies can be added to another set of reference wells.Subsequently, the labeled detection antibody specific for thatanti-PLA2R auto-antibodies is added to both sample and reference wells,e.g. an anti-IgG antibody. This is a “sandwich” ELISA assay, where theanti-PLA2R auto-antibodies is sandwich between PLA2R and an anti-IgGantibody. Since the amount of label detected is a measure of the amountof anti-PLA2R auto-antibodies present in the wells, the amounts of labeldetected in the various wells provides means for comparing the level ofthe anti-PLA2R auto-antibodies in the sample with the reference value ofthe anti-PLA2R auto-antibodies normally present in healthy subject. Forexample, if the label is colored latex beads, greater color intensity inthe sample wells compared to the reference wells indicates that thelevel of anti-PLA2R auto-antibodies in the sample is higher than twofold of the reference value of the anti-PLA2R auto-antibodies normallypresent in healthy subject. On the other hand, if the color intensity inthe sample wells is lower compared to the reference well, that indicatethat the level of the anti-PLA2R auto-antibodies in the sample is atleast two fold lower than of the reference value of the auto-antiPLA2Rantibody normally present in healthy subject.

Embodiments of the invention also provide for systems (and computerreadable media for causing computer systems) to perform a method fordiagnosing MN in a subject, assessing a subject's risk of developing MN,or monitoring treatment efficacy of a subject with MN.

In one embodiment, provided herein is a system comprising: a measuringmodule measuring auto-antibody information comprising a detectablesignal from an immunoassay indicating the presence or level ofantibodies that are reactive to a PLA2R from a sample obtained form asubject; a storage module configured to store data output from themeasuring module; a comparison module adapted to compare the data storedon the storage module with reference and/or control data, and to providea retrieved content, and an output module for displaying the retrievedcontent for the user, wherein the retrieved content the presence ofdetectable amount of antibodies reactive against PLA2R indicates thatthe subject has MN or has a relapse of MN.

In one embodiment, provided herein is a system to facilitate theprognosis evaluation of membranous nephropathy (MN) in a subject,comprising: a determination module configured to receive and outputauto-antibody information to a PLA2R from a sample obtained from asubject, wherein the auto-antibodies information measures the level ofauto antibodies that are reactive to the PLA2R; a storage moduleconfigured to store output information from the determination module; acomparison module adapted to compare the data stored on the storagemodule with reference and/or control data, and to provide a comparisoncontent, and an output module for displaying the comparison content forthe user, wherein if there is no detectable amount of auto antibodiesreactive against PLA2R then the subject is in remission or if there is areduction of at least 10% to a prior reading, then the treatment for MNis effective in the subject.

In some embodiments, the control data comprises previous data from thesame subject wherein the previous data had indicated detectable amountsof auto-antibodies.

In one embodiment, provided herein is a computer readable storage mediumcomprising: a storing data module containing data from a sample obtainedfrom a subject that represents a signal level from an immunoassay forantibodies that are reactive to a PLA2R; a comparison module thatcompares the data stored on the storing data module with a referencedata and/or control data, and to provide a comparison content, and anoutput module displaying the comparison content for the user, whereinthe presence of a detectable amount of antibodies reactive against PLA2Rof at least 10% relative to the reference data and/or control dataindicates that the subject has MN or has a relapse of MN.

In one embodiment, the control data comprises data from a population ofnon-MN healthy individuals which is the detection signal obtained usinghuman sera from non-MN healthy individuals at 1:100 dilution with 1×PBSto immuno-react with 0.5 μg of native PLA2R, wherein horse-radishperoxidase anti-human IgG antibody is the labeled detection antibody andthe detection signal is chemiluminescence.

Embodiments of the invention can be described through functionalmodules, which are defined by computer executable instructions recordedon computer readable media and which cause a computer to perform methodsteps when executed. The modules are segregated by function for the sakeof clarity. However, it should be understood that the modules/systemsneed not correspond to discreet blocks of code and the describedfunctions can be carried out by the execution of various code portionsstored on various media and executed at various times. Furthermore, itshould be appreciated that the modules may perform other functions, thusthe modules are not limited to having any particular functions or set offunctions.

The computer readable storage media #30 can be any available tangiblemedia that can be accessed by a computer. Computer readable storagemedia includes volatile and nonvolatile, removable and non-removabletangible media implemented in any method or technology for storage ofinformation such as computer readable instructions, data structures,program modules or other data. Computer readable storage media includes,but is not limited to, RAM (random access memory), ROM (read onlymemory), EPROM (eraseable programmable read only memory), EEPROM(electrically eraseable programmable read only memory), flash memory orother memory technology, CD-ROM (compact disc read only memory), DVDs(digital versatile disks) or other optical storage media, magneticcassettes, magnetic tape, magnetic disk storage or other magneticstorage media, other types of volatile and non-volatile memory, and anyother tangible medium which can be used to store the desired informationand which can accessed by a computer including and any suitablecombination of the foregoing.

Computer-readable data embodied on one or more computer-readable mediamay define instructions, for example, as part of one or more programsthat, as a result of being executed by a computer, instruct the computerto perform one or more of the functions described herein, and/or variousembodiments, variations and combinations thereof. Such instructions maybe written in any of a plurality of programming languages, for example,Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic,COBOL assembly language, and the like, or any of a variety ofcombinations thereof. The computer-readable media on which suchinstructions are embodied may reside on one or more of the components ofeither of a system, or a computer readable storage medium describedherein, may be distributed across one or more of such components.

The computer-readable media may be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the present invention discussed herein. Inaddition, it should be appreciated that the instructions stored on thecomputer-readable medium, described above, are not limited toinstructions embodied as part of an application program running on ahost computer. Rather, the instructions may be embodied as any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a computer to implement aspects of the present invention. Thecomputer executable instructions may be written in a suitable computerlanguage or combination of several languages. Basic computationalbiology methods are known to those of ordinary skill in the art and aredescribed in, for example, Setubal and Meidanis et al., Introduction toComputational Biology Methods (PWS Publishing Company, Boston, 1997);Salzberg, Searles, Kasif, (Ed.), Computational Methods in MolecularBiology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

The functional modules of certain embodiments of the invention includeat minimum a measuring module #40, a storage module #30, a comparisonmodule #80, and a output module #110. The functional modules can beexecuted on one, or multiple, computers, or by using one, or multiple,computer networks. The measuring module has computer executableinstructions to provide e.g., expression information in computerreadable form.

The measuring module #40, can comprise any system for detecting a signalrepresenting expression level of anti-PLA2R auto-antibodies. Suchsystems can include DNA microarrays, RNA expression arrays, any ELISAdetection system and/or any Western blotting detection system.

The information determined in the determination system can be read bythe storage module #30. As used herein the “storage module” is intendedto include any suitable computing or processing apparatus or otherdevice configured or adapted for storing data or information. Examplesof electronic apparatus suitable for use with the present inventioninclude stand-alone computing apparatus, data telecommunicationsnetworks, including local area networks (LAN), wide area networks (WAN),Internet, Intranet, and Extranet, and local and distributed computerprocessing systems. Storage modules also include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc storagemedia, magnetic tape, optical storage media such as CD-ROM, DVD,electronic storage media such as RAM, ROM, EPROM, EEPROM and the like,general hard disks and hybrids of these categories such asmagnetic/optical storage media. The storage module is adapted orconfigured for having recorded thereon expression level or protein levelinformation. Such information may be provided in digital form that canbe transmitted and read electronically, e.g., via the Internet, ondiskette, via USB (universal serial bus) or via any other suitable modeof communication.

As used herein, “stored” refers to a process for encoding information onthe storage module. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising expression level information.

In one embodiment the reference data stored in the storage module to beread by the comparison module is e.g., expression data obtained from apopulation of non-MN subjects, a population of MN subjects or expressiondata obtained from the same subject at a prior time point using themeasuring module #40.

The “comparison module” #80 can use a variety of available softwareprograms and formats for the comparison operative to compare expressiondata determined in the measuring module to reference samples and/orstored reference data. In one embodiment, the comparison module isconfigured to use pattern recognition techniques to compare informationfrom one or more entries to one or more reference data patterns. Thecomparison module may be configured using existingcommercially-available or freely-available software for comparingpatterns, and may be optimized for particular data comparisons that areconducted. The comparison module provides computer readable informationrelated to normalized expression level of auto-antibodies,presence/absence of MN in an individual, efficacy of treatment in anindividual, and/or method for treating an individual.

The comparison module, or any other module of the invention, may includean operating system (e.g., UNIX) on which runs a relational databasemanagement system, a World Wide Web application, and a World Wide Webserver. World Wide Web application includes the executable codenecessary for generation of database language statements (e.g.,Structured Query Language (SQL) statements). Generally, the executableswill include embedded SQL statements. In addition, the World Wide Webapplication may include a configuration file which contains pointers andaddresses to the various software entities that comprise the server aswell as the various external and internal databases which must beaccessed to service user requests. The Configuration file also directsrequests for server resources to the appropriate hardware—as may benecessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in aparticular preferred embodiment of the present invention, users candirectly access data (via Hypertext links for example) residing onInternet databases using a HTML interface provided by Web browsers andWeb servers.

The comparison module provides a computer readable comparison resultthat can be processed in computer readable form by predefined criteria,or criteria defined by a user, to provide a content-based in part on thecomparison result that may be stored and output as requested by a userusing an output module #110.

The content based on the comparison result, may be an expression valuecompared to a reference showing the presence/absence of MN in anindividual or an assessed risk of a subject to develop MN.

In one embodiment of the invention, the content based on the comparisonresult is displayed on a computer monitor #120. In one embodiment of theinvention, the content based on the comparison result is displayedthrough printable media #130, #140. The display module can be anysuitable device configured to receive from a computer and displaycomputer readable information to a user. Non-limiting examples include,for example, general-purpose computers such as those based on IntelPENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC,Hewlett-Packard PA-RISC processors, any of a variety of processorsavailable from Advanced Micro Devices (AMD) of Sunnyvale, Calif., or anyother type of processor, visual display devices such as flat paneldisplays, cathode ray tubes and the like, as well as computer printersof various types.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content based on the comparison result. Itshould be understood that other modules of the invention can be adaptedto have a web browser interface. Through the Web browser, a user mayconstruct requests for retrieving data from the comparison module. Thus,the user will typically point and click to user interface elements suchas buttons, pull down menus, scroll bars and the like conventionallyemployed in graphical user interfaces.

The present invention therefore provides for systems (and computerreadable media for causing computer systems) to perform methods fordiagnosing MN or assessing treatment prognosis of MN in an individual.

Systems and computer readable media described herein are merelyillustrative embodiments of the invention for detecting anti-PLA2Rautoantibodies in an individual, and are not intended to limit the scopeof the invention. Variations of the systems and computer readable mediadescribed herein are possible and are intended to fall within the scopeof the invention.

The modules of the machine, or those used in the computer readablemedium, may assume numerous configurations. For example, function may beprovided on a single machine or distributed over multiple machines.

Sample Collection and Preparation

Collections of samples can be performed by methods well known to thoseskilled in the art.

For example, the patient's blood can be drawn by trained medicalpersonnel directly into anti-coagulants such as citrate and EDTA. Thewhole blood can be separated into the plasma portion, the cells, andplatelets portion by refrigerated centrifugation at 3500×G for 2minutes. After centrifugation, the supernatant is the plasma.

Alternately, the serum can be collected from the whole blood. Collectthe blood in a hard plastic or glass tube; blood will not clot in softplastic. Draw 15 mL of whole blood for 6 mL of serum. The whole blood isallowed to stand at room temperature for 30 minutes to 2 hours until aclot has formed. Carefully separate clot from the sides of the containerusing a glass rod or wooden applicator stick and leave overnight at 4°C. After which, decant serum, centrifuge, and/or using a Pasteurpipette, remove serum into a clean tube. Clarify the serum bycentrifugation at 2000-3000 rpm for 10 minutes. The serum is stored at−20° or −80° C. before analysis for auto-antibodies against PLA2R isperformed. Detailed description of obtaining serum using collectiontubes can be found in U.S. Pat. No. 3,837,376 and is hereby incorporatedby reference in it entirety. Blood collection tubes can also bepurchased from BD Diagnostic Systems, Greiner Bio-One, and KendallCompany.

Detection of PLA2R Antibodies

The detection of auto-antibodies against human or pig PLA2R in blood,serum or plasma can be detected by any method known in the art.Preferably by ELISA, wherein the detection method is an immunochemicalmethod involving the binding of the auto-antibodies with a PLA2R proteinor fragments thereof. Formation of the antibody-protein complex is thendetected by a variety of methods known in the art.

Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassayor EIA, is a biochemical technique used mainly in immunology to detectthe presence of an antibody or an antigen in a sample. The ELISA hasbeen used as a diagnostic tool in medicine and plant pathology, as wellas a quality control check in various industries. For the methodsdescribed herein, in the ELISA a known amount of antigen (PLA2R orfragments thereof) is affixed to a surface, and then the sample, e.g.blood, serum or plasma, suspected of containing auto-antibodies toPLA2R, is washed over the surface so that the auto-antibodies can bindto the immobilized antigen. The surface is washed to remove any unboundprotein and a detection antibody is applied to the surface. Thedetection antibody is specific to antibodies from the subject. Forexample, if the subject is a human, the detection antibody should be ananti-human IgG antibody. If the subject is a dog, the detection antibodythen should an anti-dog IgG antibody. This detection antibody is linkedto an enzyme, and in the final step a substance is added that the enzymecan convert to some detectable signal. For example, in the case offluorescence ELISA, when light is shone upon the sample, anyantigen/antibody complexes will fluoresce so that the amount ofantibodies in the sample can be measured. This is the indirectenzyme-linked immunosorbent assay. A schematic diagram of the indirectELISA is shown in FIG. 7.

The following is a general standard protocol for setting up andperforming an indirect enzyme-linked immunosorbent assay. Using 96-wellmicrotiter plates (Falcon Pro-Bindassay plate 3915; Becton Dickinson,Paramus, N.J.), test wells are coated with antigen (PLA2R or fragmentsthereof) by incubation with 100 μl of purified PLA2R (3 μg/ml in PBS)per well overnight at room temperature, with PBS substituted for theantigen in control wells. After the plates have been washed three timeswith PBS-Tween, 250 μl of 2% BSA in PBS is added to each well, and theplates are incubated for 1 h at room temperature. The plates are washedthree times with PBS-Tween and incubated for 1 h at room temperaturewith test sera and control sera (one high-positive serum specimen, twonegative serum specimens, and one weak-positive serum specimen) diluted1:100 in PBS-Tween-BSA; each serum specimen is tested in triplicate inantigen-coated wells as well as in antigen control wells. The plate isthen assayed (with appropriate controls) for the presence of humanauto-antibodies IgG against PLA2R by incubation for 1 h at roomtemperature with 100 μl of goat anti-human IgG conjugated withhorseradish peroxidase (Bio-Rad, Richmond, Calif.) per well diluted1:2,000 in PBS-Tween-BSA. After three washes in PBS-Tween, the substratesolution (o-phenylenediamine dihydrochloride; Sigma) is added to eachwell. The plates are then incubated for 30 min at room temperature indarkness, and the reaction is terminated by the addition of 2N sulfuricacid. The optical density values at 490 nm (OD₄₉₀) are measured in amicro plate ELISA reader. For each serum specimen, mean OD₄₉₀ readingsare calculated for the test wells and for the antigen control wells, thelatter being subtracted from the former to obtain the net ELISA value.

Performing an ELISA involves at least one antibody with specificity fora particular antigen. A known amount of antigen (PLA2R) is immobilizedon a solid support (usually a polystyrene micro titer plate) eithernon-specifically (via adsorption to the surface) or specifically (viacapture by another antibody specific to the same antigen, in a“sandwich” ELISA). After the antigen is immobilized, the detectionantibody is added, forming a complex with the antigen. The detectionantibody can be covalently linked to an enzyme, or can itself bedetected by a secondary antibody which is linked to an enzyme throughbio-conjugation. Between each step the plate is typically washed with amild detergent solution to remove any proteins or antibodies that arenot specifically bound. After the final wash step the plate is developedby adding an enzymatic substrate to produce a visible signal, whichindicates the quantity of antigen in the sample. Older ELISAs utilizechromogenic substrates, though newer assays employ fluorogenicsubstrates with much higher sensitivity.

In another embodiment, a competitive ELISA is used. Purified anti-PLA2Rantibodies that are not derived from the subject are coated on the solidphase of multi-wells. Serum sample recombined PLA2R, (the antigen) orfragments thereof and horseradish peroxidase labeled with anti-PLA2Rantibodies (conjugated) are added to coated wells, and form competitivecombination. After incubation, if the auto-antibody level against PLA2Rcontent is high in the sample, a complex ofPLA2R-auto-antibodies-anti-PLA2R labeled with HRP will form. Wash wellswill remove the complex, and incubate with TMB(3,3′,5,5′-tetramethylbenzidene) color development substrate forlocalization of horseradish peroxidase-conjugated antibodies in thewells. Subsequently there will be no color change or little colorchange. If there are no auto-antibodies against PLAR2 in the serumsample, there will be much color change. Such a competitive ELSA test isspecific, sensitive, reproducible and easy to operate.

In one embodiment, the reverse-sandwich (RS) ELISA is used (Miyazawa H.,et. al, J Allergy Clin Immunol. 1988; 82:407-413), wherein the antibodyof interest, in the methods described herein, the auto-antibodiesagainst PLA2R, is sandwiched by antigens (PLA2R): one antigen is affixedto a surface and the second antigen is soluble and tagged. This methodis also known as the double-antigen sandwich method. A schematic diagramof the RS ELISA is shown in FIG. 7.

The following is a general standard protocol for setting up andperforming a RS-ELISA. A 0.1-ml quantity of PLA2R (0.3 μg/ml) or PLA2R(0.9 μg/ml) plus bovine serum albumin (BSA; 25 μg/ml) in 0.5 M NaCl-0.1%NaN₃-0.05 M sodium carbonate (pH 9.6) is added to wells of Maxisorpmicroplates (Nalge Nunc, Copenhagen, Denmark). The plates are incubatedovernight at 4° C. for antigen immobilization. After the wells arewashed test sera diluted 1:4, 1:40, and 1:400 with FBS-PBST (10%[vol/vol] fetal bovine serum [FBS], 0.1% NaN₃-phosphate-buffered saline[PBS]-0.05% Tween-20 [PBST]) are added, and the plates are incubated for60 min at room temperature. Seven threefold serial dilutions of thereference serum are used. After another wash, biotinylated PLA2R orPLA2R (0.05 μg/ml) in FBS-PBST is then added to the wells, and thereaction is allowed to take place for 60 min at room temperature. Thewells are washed again, streptavidin-conjugated β-d-galactosidase (GIBCOBRL, Life Technologies Inc., Rockville, Md.; diluted 1:50,000 in PBSTcontaining 1% BSA) is added, and the plates are incubated for 60 min atroom temperature. After another wash, 0.2 mM4-methylumbelliferyl-β-d-galactoside (Sigma Chemical Co., St. Louis,Mo.) in 0.1 M NaCl-1 mM MgCl₂-0.1% BSA-0.1% NaN₃-0.01 M sodium phosphate(pH 7.0) is added. The wells are sealed with tape, and the plates areimmersed in 37° C. water for 60 min. Finally, 0.1 ml of 0.1 Mglycine-NaOH (pH 10.2) is added to each well to stop the enzymereaction. The fluorescence units (FU) in each well is measured with aFluoroskan II apparatus (Flow Laboratories, Rockville, Md.). Theantibody concentrations of the test sera are calculated from thetitration curve of the reference serum with known antibody units permilliliter.

In one preferred embodiment, the detection antibody is detectablylabeled by linking the antibody to an enzyme. The enzyme, in turn, whenexposed to its substrate, will react with the substrate in such a manneras to produce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or by visual means. Enzymes which canbe used to detectably label the antibodies of the present inventioninclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

In other embodiments, the detection antibody is label with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are CY dyes, fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

A detection antibody can also be detectably labeled using fluorescenceemitting metals such as 152Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

A detection antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-antibodyis then determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of particularlyuseful chemiluminescent labeling compounds are luminol, luciferin,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

There are other different forms of ELISA, which are well known to thoseskilled in the art. The standard techniques known in the art for ELISAare described in “Methods in Immunodiagnosis”, 2nd Edition, Rose andBigazzi, eds. John Wiley & Sons, 1980; Campbell et al., “Methods andImmunology”, W. A. Benjamin, Inc., 1964; and Oellerich, M. 1984, J.Clin. Chem. Clin. Biochem. 22:895-904.

Other techniques can be used to detect PLA2R auto-antibodies in asample. One such technique is Western blotting (Towbin et at., Proc.Nat. Acad. Sci. 76:4350 (1979)), another is an adaptation of the Westernblot, the dot blots. In the Western blots, the PLA2R protein orfragments thereof can be dissociated with detergents and heat, and runon an SDS-PAGE gel before being transferred to a solid support, such asa nitrocellulose filter. The filter is washed with a sample suspected ofcontaining auto-antibodies against PLA2R. The filter is then washed toremove unbound proteins and proteins with non-specific binding.Detectably labeled enzyme-linked secondary antibodies can then be usedto detect and assess the amount of auto-antibodies in the sample tested.The intensity of the signal from the detectable label corresponds to theamount of enzyme present, and therefore the amount of auto-antibodiesagainst PLA2R. Levels can be quantified, for example by densitometry.

Another immunological assay is nephelometric immunoassays. Nephelometricimmunoassays are known to one skilled in the art and can be performed tothe methods as described in U.S. Pat. Nos. 4,730,922, 4,268,171,4,401,387, 4,408,880, 4,889,815, 4,690,906, 4,784,947, and 516,223, andall of which are hereby incorporated by reference in their entirety.

As positive control of antibodies against PLA2R, a known quantity ofanti-PLA2R antibodies can be used. The anti-PLA2R antibodies can beobtained from commercial source such as INVITROGEN Inc., MILLIPORE,SIGMA-ALDRICH, R&D Systems, ABCAM and the World's Antibody Gateway (freesearch engine of over 150 antibody companies) and GeneTexto name a few.The antibodies can be polyclonal or monoclonal antibodies.Alternatively, antibodies can be raised against the human PLA2R protein(GENBANK™ Accession No. NP_(—)001007268; SEQ. ID. NO. 1 andNP_(—)031392.3, SEQ. ID. NO. 2) or fragments thereof by one of skill inthe art. Methods for the production of antibodies are disclosed in PCTpublication WO 97/40072 or U.S. Application. No. 2002/0182702, which areherein incorporated by reference. The processes of immunization toelicit antibody production in a mammal, the generation of hybridomas toproduce monoclonal antibodies, and the purification of antibodies may beperformed by described in “Current Protocols in Immunology” (CPI) (JohnWiley and Sons, Inc.) and Antibodies: A Laboratory Manual (Ed Harlow andDavid Lane editors, Cold Spring Harbor Laboratory Press 1988) which areboth incorporated by reference herein in their entireties.

The detection of auto-antibodies against PLA2R is considered positivewhen the immunoassay signal is at least 10% over that of the controlimmunoassay signal in the absence of an antibody against the PLA2R orfragments thereof or in the presence of a non-related, non-PLA2R bindingantibody. In another embodiment, the control immunoassay signal is thatobtained with the serum of non-MN healthy subject, thes subjects do nothave the clinical features of the disease. In another embodiment, thecontrol immunoassay signal is the average value obtained for apopulation of non-MN healthy subjects. A population is at least 25non-MN healthy subjects, preferably more. The increase is at least 5%,at least 10%, at least 20%, at least 30%, at least 50%, at least 100%,at least 200%, at least 300%, at least 500%, at least 1000%, or more andincluding all the percentages between 10-1000%.

In one embodiment, detection of auto-antibodies comprises identifyingand detecting elevated amount of the mRNA that codes for the antibodies.There are many methods of detecting, identifying and determining mRNAthat are well known in the art, e.g. Northern blots and RT-PCR. In oneembodiment, the mRNA of can be determined by quantitative real-time PCR.Real time PCR is an amplification technique that can be used todetermine levels of mRNA expression. (See, e.g., Gibson et al., GenomeResearch 6:995-1001, 1996; Heid et al., Genome Research 6:986-994,1996). Real-time PCR evaluates the level of PCR product accumulationduring amplification. This technique permits quantitative evaluation ofmRNA levels in multiple samples. For mRNA levels, mRNA is extracted froma biological sample, e.g. a blood sample, and cDNA is prepared usingstandard techniques. Real-time PCR can be performed, for example, usinga Perkin Elmer/Applied Biosystems (Foster City, Calif.) 7700 Prisminstrument. Matching primers and fluorescent probes can be designed forgenes of interest using, for example, the primer express programprovided by Perkin Elmer/Applied Biosystems (Foster City, Calif.).Optimal concentrations of primers and probes can be initially determinedby those of ordinary skill in the art, and control (for example,beta-actin) primers and probes can be obtained commercially from, forexample, Perkin Elmer/Applied Biosystems (Foster City, Calif.). Toquantify the amount of the specific nucleic acid of interest in asample, a standard curve is generated using a control. Standard curvescan be generated using the Ct values determined in the real-time PCR,which are related to the initial concentration of the nucleic acid ofinterest used in the assay. Standard dilutions ranging from 10-106copies of the gene of interest are generally sufficient. In addition, astandard curve is generated for the control sequence. This permitsstandardization of initial content of the nucleic acid of interest in atissue sample to the amount of control for comparison purposes.

Methods of real-time quantitative PCR using TaqMan probes are well knownin the art. Detailed protocols for real-time quantitative PCR areprovided, for example, for RNA in: Gibson et al., 1996, Genome Res.,10:995-1001; and for DNA in: Heid et al., 1996, Genome Res., 10:986-994.

The TaqMan based assays use a fluorogenic oligonucleotide probe thatcontains a 5′ fluorescent dye and a 3′ quenching agent. The probehybridizes to a PCR product, but cannot itself be extended due to ablocking agent at the 3′ end. When the PCR product is amplified insubsequent cycles, the 5′ nuclease activity of the polymerase, forexample, AmpliTaq, results in the cleavage of the TaqMan probe. Thiscleavage separates the 5′ fluorescent dye and the 3′ quenching agent,thereby resulting in an increase in fluorescence as a function ofamplification (see, for example, at the Perkin Elmer World Wide Web).

In another embodiment, the detection of RNA transcripts can be achievedby Northern blotting, wherein a preparation of RNA is run on adenaturing agarose gel, and transferred to a suitable support, such asactivated cellulose, nitrocellulose or glass or nylon membranes. Labeled(e.g., radiolabeled) cDNA or RNA is then hybridized to the preparation,washed and analyzed by methods such as autoradiography.

In another embodiment, the detection of RNA transcripts can further beaccomplished using known amplification methods. For example, it iswithin the scope of the present invention to reverse transcribe mRNAinto cDNA followed by polymerase chain reaction (RT-PCR); or, to use asingle enzyme for both steps as described in U.S. Pat. No. 5,322,770, orreverse transcribe mRNA into cDNA followed by symmetric gap lipase chainreaction (RT-AGLCR) as described by R. L. Marshall, et al., PCR Methodsand Applications 4: 80-84 (1994). One suitable method for detectingenzyme mRNA transcripts is described in reference Pabic et. al.Hepatology, 37(5): 1056-1066, 2003, which is herein incorporated byreference in its entirety.

In other embodiments, the detection of RNA transcripts can be achievedwith other known amplification methods which include but are not limitedto the so-called “NASBA” or “3SR” technique described in PNAS USA 87:1874-1878 (1990) and also described in Nature 350: 91-92 (1991); Q-betaamplification as described in published European Patent Application(EPA) No. 4544610; strand displacement amplification (as described in G.T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European PatentApplication No. 684315; and target mediated amplification, as describedby PCT Publication WO 9322461.

Encompassed in the method described herein is employing in situhybridization visualization for the detection of auto-antibodies toPLA2R RNA transcripts in blood samples. In in situ hybridization, aradioactively labeled antisense RNA probe is hybridized with a thinsmear of platelets, after which the smear of platelets is washed,cleaved with RNase, and exposed to a sensitive emulsion forautoradiography. The samples can be stained with haematoxylin todemonstrate the histological composition of the sample, and dark fieldimaging with a suitable light filter shows the developed emulsion.Non-radioactive labels such as digoxigenin can also be used.

Alternatively, mRNA expression can be detected on a DNA array, chip or amicroarray. Oligonucleotides corresponding to auto-antibodies to PLA2RRNA transcripts are immobilized on a chip which is then hybridized withlabeled nucleic acids of a sample of platelets obtained from a patient.Positive hybridization signal is obtained with a sample containingauto-antibodies to PLA2R RNA transcripts. Methods of preparing DNAarrays and their use are well known in the art. (See, for example U.S.Pat. Nos. 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S.20030157485 and Schena et al. 1995 Science 20:467-470; Gerhold et al.1999 Trends in Biochem. Sci. 24, 168-173; and Lennon et al. 2000 Drugdiscovery Today 5: 59-65, which are herein incorporated by reference intheir entirety). Serial Analysis of Gene Expression (SAGE) can also beperformed (See for example U.S. Patent Application 20030215858).

To monitor mRNA levels, for example, mRNA is extracted from the bloodsample to be tested, reverse transcribed, and fluorescent-labeled cDNAprobes are generated. The microarrays capable of hybridizing to cDNA arethen probed with the labeled cDNA probes, the slides scanned andfluorescence intensity measured. This intensity correlates with thehybridization intensity and expression levels. The cDNAs correspond tothe auto-antibodies to PLA2R RNA transcripts, particularly in thevariable region of the antibody.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that can be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided, for example, in Innis et al. (1990) PCR Protocols, A Guide toMethods and Applications, Academic Press, Inc. N.Y.

Recombinant PLA2R Protein and PLA2R Expression Vectors

Recombinant PLA2R protein and fragments thereof can also be synthesizedand purified by molecular methods that are well known in the art. Forexample, recombinant proteins can be expressed in bacteria, mammal,insect, yeast, or plant cells.

Conventional polymerase chain reaction (PCR) cloning techniques can beused to clone a nucleic acid encoding a PLA2R, using the mRNA of thePLA2R as the template for PCR Cloning. In some embodiments, the mRNAtemplates of the human PLA2R are Genbank Accession Nos. NM_(—)001007267,SEQ. ID. NO. 3 and NM_(—)007366.3, SEQ. ID. NO. 4. Ideally, restrictionenzyme digestion recognition sites should be designed at the ends of thesense and anti-sense strand of the PCR primers to facilitate ligation ofthe amplified nucleic acid into a cloning vector or other vectors.Alternatively, a 3′-A overhang can be include for the purpose ofTA-cloning that is well known in the art. Such coding nucleic acids with3′A overhangs can be easily ligated into the Invitrogentopoisomerase-assisted TA vectors such as pCR®-TOPO, pCR®-Blunt II-TOPO,pENTR/D-TOPO®, and pENTR/SD/D-TOPO®. The coding nucleic acid can becloned into a general purpose cloning vector such as pUC19, pBR322,pBLUESCRIPT vectors (STRATAGENE Inc.) or pCR TOPO® from Invitrogen Inc.The resultant recombinant vector carrying the nucleic acid encoding aPLA2R can then subcloned into protein expression vectors or viralvectors for the synthesis of PLA2R fusion protein in a variety ofprotein expression systems using host cells selected from the groupconsisting of mammalian cell lines, insect cell lines, yeast, bacteria,and plant cells. Protease cleavage sites can also be designed andincluded within the nucleic acid to facilitate the liberation of PLA2Rfrom a larger fusion protein, e.g. His-PLA2R or thioredoxin-PLA2R.Examples of protease cleavage sites include but are not limited to thoseof enterokinase, chymotrypsin, and thrombin.

PCR amplified coding nucleic acids can be cloned into a vector using theTOPO® cloning method in Invitrogen topoisomerase-assisted TA vectorssuch as pCR®-TOPO, pCR® Blunt II-TOPO, pENTR/D-TOPO®, andpENTR/SD/D-TOPO®. Both pENTR/D-TOPO®, and pENTR/SD/D-TOPO® aredirectional TOPO entry vectors which allow the cloning of the DNAsequence in the 5′→3′ orientation into a GATEWAY® expression vector.Directional cloning in the 5′→3′ orientation facilitate theunidirectional insertion of the DNA sequence into a protein expressionvector such that the promoter is upstream of the 5′ ATG start codon ofthe nucleic acid, thus enabling promoter-driven protein expression. Therecombinant vector carrying a PLA2R coding nucleic acid can betransfected into and propagated in a general cloning E. coli cells suchas XL1Blue, SURE (STRATAGENE) and TOP-10 cells (INVITROGEN).

Different expression vectors are available for the expression andpurification of a recombinant protein produced from a heterologousprotein expression system can be made. Heterologous protein expressionsystems that use host cells selected from, e.g., mammalian, insect,yeast, bacterial, or plant cells are well known to one skilled in theart. The expression vector should have the necessary 5′ upstream and 3′downstream regulatory elements such as promoter sequences, ribosomerecognition and binding TATA box, and 3′ UTR AAUAAA (SEQ. DI. NO. 5)transcription termination sequence for efficient gene transcription andtranslation in its respective host cell. The expression vector may haveadditional sequence such as 6X-histidine, V5, thioredoxin,glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose bindingpeptide, metal-binding peptide, HA and “secretion” signals (Honeybeemelittin, α-factor, PHO, Bip), which are incorporated into the expressedrecombinant protein. In addition, there can be enzyme digestion sitesincorporated after these sequences to facilitate enzymatic removal ofadditional sequence after they are not needed. These additionalsequences are useful for the detection of recombinant proteinexpression, for protein purification by affinity chromatography,enhanced solubility of the recombinant protein in the host cytoplasm,for better protein expression especially for small protein fragmentsand/or for secreting the expressed recombinant protein out into theculture media, into the periplasm of the prokaryote bacteria, or to thespheroplast of yeast cells. The expression of recombinant protein can beconstitutive in the host cells or it can be induced, e.g., with coppersulfate, sugars such as galactose, methanol, methylamine, thiamine,tetracycline, infection with baculovirus, and(isopropyl-beta-D-thiogalactopyranoside) IPTG, a stable synthetic analogof lactose.

In some embodiments, recombinant PLA2R can be expressed in a variety ofexpression host cells e.g., bacteria, such as E. coli, yeast, mammalian,insect, and plant cells such as Chlamydomonas, or even from cell-freeexpression systems. From a cloning vector, the nucleic acid can besubcloned into a recombinant expression vector that is appropriate forthe expression of the protein in mammalian, insect, yeast, bacterial, orplant cells or a cell-free expression system such as a rabbitreticulocyte expression system. Subcloning can be achieved by PCRcloning, restriction digestion followed by ligation, or recombinationreaction such as those of the lambda phage-based site-specificrecombination using the Gateway® LR and BP CLONASE™ enzyme mixtures.Subcloning should be unidirectional such that the 5′ ATG start codon ofthe nucleic acid is downstream of the promoter in the expression vector.Alternatively, when the coding nucleic acid is cloned intopENTR/D-TOPO®, pENTR/SD/D-TOPO® (directional entry vectors), or any ofthe Invitrogen's Gateway® Technology pENTR (entry) vectors, the codingnucleic acid can be transferred into the various GATEWAY® expressionvectors (destination) for protein expression in mammalian cells, E.coli, insects and yeast respectively in one single recombinationreaction. Some of the GATEWAY® destination vectors are designed for theconstructions of baculovirus, adenovirus, adeno-associated virus (AAV),retrovirus, and lentiviruses, which upon infecting their respective hostcell, permit heterologous expression of the recombinant protein in thehost cells. Transferring a gene into a destination vector isaccomplished in just two steps according to manufacturer's instructions.There are GATEWAY® expression vectors for protein expression in E. coli,insect cells, mammalian cells, and yeast. Following transformation andselection in E. coli, the expression vector is ready to be used forexpression in the appropriate host.

Examples of other expression vectors and host cells are the pET vectors(NOVAGEN), pGEX vectors (Amersham Pharmacia), and pMAL vectors (NewEngland labs. Inc.) for protein expression in E. coli host cells such asBL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3)(NOVAGEN); the strong CMV promoter-based pcDNA3.1 (INVITROGEN) andpCIneo vectors (Promega) for expression in mammalian cell lines such asCHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviralvector vectors pADENO X, pAd5F35, pLP-ADENO-X-CMV (CLONTECH),pAd/CMV/V5-DEST, pAd-DEST vector (INVITROGEN) for adenovirus-mediatedgene transfer and expression in mammalian cells; pLNCX2, pLXSN, andpLAPSN retrovirus vectors for use with the RETRO-X™ system from Clontechfor retroviral-mediated gene transfer and expression in mammalian cells;pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ(INVITROGEN) for lentivirus-mediated gene transfer and expression inmammalian cells; adenovirus-associated virus expression vectors such aspAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) foradeno-associated virus-mediated gene transfer and expression inmammalian cells; BACpak6 baculovirus (CLONTECH) and pFASTBAC™ HT(INVITROGEN) for the expression in Spodopera frugiperda 9 (Sf9) and Sf11insect cell lines; pMT/BiP/V5-His (INVITROGEN) for the expression inDrosophila Schneider S2 cells; Pichia expression vectors pPICZα, pPICZ,pFLDα and pFLD (Invitrogen) for expression in Pichia pastoris andvectors pMETα and pMET for expression in P. methanolica; pYES2/GS andpYD1 (INVITROGEN) vectors for expression in yeast Saccharomycescerevisiae. Recent advances in the large scale expression heterologousproteins in Chlamydomonas reinhardtii are described by Griesbeck C. et.al. 2006 Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods MolMed. 94:191-5. Foreign heterologous coding sequences are inserted intothe genome of the nucleus, chloroplast and mitochodria by homologousrecombination. The chloroplast expression vector p64 carrying theversatile chloroplast selectable marker aminoglycoside adenyltransferase (aadA), which confers resistance to spectinomycin orstreptomycin, can be used to express foreign protein in the chloroplast.The biolistic gene gun method can be used to introduce the vector in thealgae. Upon its entry into chloroplasts, the foreign DNA is releasedfrom the gene gun particles and integrates into the chloroplast genomethrough homologous recombination.

Recombinant protein expression in the different host cells can beconstitutive or inducible with inducers such as copper sulfate, sugarssuch as galactose, methanol, methylamine, thiamine, tetracycline, orIPTG. After the protein is expressed in the host cells, the host cellsare lysed to liberate the expressed protein for purification. Methods oflysing the various host cells are featured in “Sample Preparation-Toolsfor Protein Research” EMD Bioscience and in the Current Protocols inProtein Sciences (CPPS). A preferred purification method is affinitychromatography such as ion-metal affinity chromatograph using nickel,cobalt, or zinc affinity resins for histidine-tagged recombinantprotein. Methods of purifying histidine-tagged recombinant proteins aredescribed by CLONTECH using their TALON® cobalt resin and by NOVAGEN intheir pET system manual, 10th edition. Another preferred purificationstrategy is by immuno-affinity chromatography, for example, anti-mycantibody conjugated resin can be used to the affinity purify myc-taggedrecombinant peptide. Enzymatic digestion with serine proteases such asthrombin and enterokinase cleave and release the recombinant proteinfrom the histidine or myc tag, releasing the recombinant protein fromthe affinity resin while the histidine-tags and myc-tags are leftattached to the affinity resin.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. An example of a cell-free translation systemcapable of producing proteins in high yield is described by Spirin A S.et. al., Science 242:1162 (1988). The method uses a continuous flowdesign of the feeding buffer which contains amino acids, adenosinetriphosphate (ATP), and guanosine triphosphate (GTP) throughout thereaction mixture and a continuous removal of the translated polypeptideproduct. The system uses E. coli lysate to provide the cell-freecontinuous feeding buffer. This continuous flow system is compatiblewith both prokaryotic and eukaryotic expression vectors. As an example,large scale cell-free production of the integral membrane protein EmrEmultidrug transporter is described by Chang G. el. al., Science310:1950-3 (2005).

Other commercially available cell-free expression systems include theEXPRESSWAY™ Cell-Free Expression Systems (Invitrogen) which utilize anE. coli-based in-vitro system for efficient, coupled transcription andtranslation reactions to produce up to milligram quantities of activerecombinant protein in a tube reaction format; the Rapid TranslationSystem (RTS) (Roche Applied Science) which also uses an E. coli-basedin-vitro system; and the TNT Coupled Reticulocyte Lysate Systems(Promega) which uses a rabbit reticulocyte-based in-vitro system.

Encompassed in the methods described herein is a mammalian PLA2R that ispurified from a mammal, e.g. a pig or a rabbit. In one embodiment, thenative (non-recombinant) mammalian PLA2R is purified from the kidneys exvivo. Methods of native protein purification are well known to oneskilled in the art.

Therapeutic/Prophylactic Compositions and Administration

In one embodiment, the invention provides a pharmaceutical compositioncomprising a PLA2R or fragment thereof and a pharmaceutically acceptablevehicle. The pharmaceutical composition can be a combination offull-length PLA2R and fragments of various sizes, and a pharmaceuticallyacceptable vehicle. Examples of fragments are fragments comprising theCTLDs or CRDs 4, 5 6 of PLA2R or other fragments of the extracellulardomain of PLA2R. The pharmaceutical composition is used for thetreatment of MN that is characterized by the presence of auto-antibodiesagainst PLA2R.

In one embodiment, the term “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the therapeuticis administered. Such pharmaceutical carriers can be sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations, and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co.,1990). In one embodiment, other ingredients can be added topharmaceutical formulations, including antioxidants, e.g., ascorbicacid; low molecular weight (less than about ten residues) polypeptides,e.g., polyarginine or tripeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; and sugar alcohols such asmannitol or sorbitol.

In an embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition can also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol,histidine, procaine, to name a few.

Various delivery systems are known in the art and can be used toadminister a PLA2R protein or fragments thereof, e.g., encapsulation inliposomes, microparticles, and microcapsules (see, e.g., Wu and Wu, J.Biol. Chem., 262:4429-4432 (1987)). The composition can be delivered ina vesicle, in particular a liposome (see, Langer, Science, 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler, eds. (Liss, New York 1989), pp.353-365; Lopez-Berestein, ibid., pp. 317-327; see, generally, ibid.).Methods of introduction include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compositions can be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and can be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it can be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection can be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Omcana reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

In one embodiment, the pharmaceutical formulation to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). The pH of the pharmaceutical formulationtypically should be about from 6 to 8.

In one embodiment, the composition can be delivered in a controlledrelease system. In one embodiment, a pump can be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:201 (1987); Buchwald etal., Surgery, 88:507 (1980); Saudek et al., N. Engl. J. Med., 321:574(1989)). In another embodiment, polymeric materials can be used (see,Medical Applications of Controlled Release, Langer and Wise, eds. (CRCPress, Boca Raton, Fla. 1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball, eds. (Wiley, New York1984); Ranger and Peppas, Macromol. Sci. Rev. Macromol. Chem., 23:61(1983); see also Levy et al., Science, 228:190 (1985); During et al.,Ann. Neurol., 25:35 1 (1989); Howard et al., J. Neurosurg., 7 1:105(1989)). Other controlled release systems are discussed in the review byLanger (Science, 249:1527-1533 (1990)). For examples of sustainedrelease compositions, see U.S. Pat. No. 3,773,919, EP 58,481A, U.S. Pat.No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman etal., Biopolymers 22:547 (1983) and R. Langer et al., Chem. Tech. 12:98(1982).

The precise dose to be employed in the formulation will also depend onthe route of administration, and the severity of MN and the titer ofauto-antibodies against PLA2R in the serum, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kgof the patient's body weight. Preferably, the dosage administered to apatient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight,more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Forgene therapy, viral vector should be in the range of 1×10⁶ to 10¹⁴ viralvector particles per application per patient.

In addition, in vitro or in vivo assays can optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employedwill also depend on the route of administration, and the seriousness ofthe condition being treated and should be decided according to thejudgment of the practitioner and each subject's circumstances in viewof, e.g., published clinical studies. Suitable effective dosage amounts,however, range from about 10 micrograms to about 5 grams about every 4hour, although they are typically about 500 mg or less per every 4hours. In one embodiment the effective dosage is about 0.01 mg, 0.5 mg,about 1 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about900 mg, about 1 g, about 1.2 g, about 1.4 g, about 1.6 g, about 1.8 g,about 2.0 g, about 2.2 g, about 2.4 g, about 2.6 g, about 2.8 g, about3.0 g, about 3.2 g, about 3.4 g, about 3.6 g, about 3.8 g, about 4.0 g,about 4.2 g, about 4.4 g, about 4.6 g, about 4.8 g, or about 5.0 g,every 4 hours. Equivalent dosages may be administered over various timeperiods including, but not limited to, about every 2 hours, about every6 hours, about every 8 hours, about every 12 hours, about every 24hours, about every 36 hours, about every 48 hours, about every 72 hours,about every week, about every two weeks, about every three weeks, aboutevery month, and about every two months. The effective dosage amountsdescribed herein refer to total amounts administered. The compositionscomprising PLA2R protein, fragments thereof, or expression vectorsand/or viral vectors are suitably administered to the patient at onetime or over a series of treatments. For purposes herein, a“therapeutically effective amount” of a composition comprising PLA2Rprotein, fragments thereof, or expression vectors and/or viral vectorsis an amount that is effective to reduce the amount of auto-antibodiesagainst PLA2R in a sample from a subject. The amount reduction is atleast 10% reduction in the auto-antibodies compared to the amount ofauto-antibodies present in the serum prior to the start of a treatment.

In an embodiment, the composition comprising a PLA2R or fragmentsthereof is administered in combination with immunosuppressive therapiesincluding, but not limited to, azathioprine, infliximab, omalizumab,daclizumab, adalimumab, eculizumab, efalizumab, natalizumab, andomalizumab. In another embodiment, the composition comprising a PLA2R orfragments thereof is administered in combination with immunosuppressivetherapies and cyclophosphamide, chlorambucil, and/or rituximab.

Gene Therapy

In one embodiment, the PLA2R protein or fragments thereof isadministered to an individual by any one of several gene therapytechniques known to those of skill in the art. In general, gene therapycan be accomplished by either direct transformation of target cellswithin the mammalian subject (in vivo gene therapy) or transformation ofcells in vitro and subsequent implantation of the transformed cells intothe mammalian subject (ex vivo gene therapy). A viral vector carries anucleic acid encoding PLA2R protein or fragments thereof under a tissuespecific regulatory element is administered to an individual. The tissuespecific regulatory element allows the expression of the PLA2R proteinor fragments thereof in the target cells, for example, the muscles.

The principles of gene therapy are disclosed by Oldham, R. K. (In:Principles of Biotherapy, Raven Press, N.Y., 1987), and similar texts.Disclosures of the methods and uses for gene therapy are provided byBoggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E. M. (Biol.Reprod. 42:39-49 (1990)); Ledley, F. D., In: Biotechnology, AComprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc.NY, pp 399-458 (1989)), all of which references are incorporated hereinby reference.

The nucleic acid encoding PLA2R protein or fragments thereof can beintroduced into the somatic cells of an animal (particularly mammalsincluding humans) in gene therapy. Most preferably, viral or retroviralvectors are employed for as the transfer vehicle this purpose. The genetherapy virus can be in the form of an adenovirus, adeno-associatedvirus or lentivirus.

Retroviral vectors are a common mode of delivery and in this context areretroviruses from which all viral genes have been removed or altered sothat no viral proteins are made in cells infected with the vector. Viralreplication functions are provided by the use of retrovirus “packaging”cells that produce all of the viral proteins but that do not produceinfectious virus.

Introduction of the retroviral vector DNA into packaging cells resultsin production of virions that carry vector RNA and can infect targetcells, but such that no further virus spread occurs after infection. Todistinguish this process from a natural virus infection where the viruscontinues to replicate and spread, the term transduction rather thaninfection is often used.

In one embodiment, the method of treating MN described herein provides arecombinant lentivirus for the delivery and expression of a PLA2Rprotein or fragments thereof in either dividing and non-dividingmammalian cells. The HIV-1 based lentivirus can effectively transduce abroader host range than the Moloney Leukemia Virus (MoMLV)-baseretroviral systems. Preparation of the recombinant lentivirus can beachieved using the pLenti4N5-DEST™, pLenti6/V5-DEST™ or pLenti vectorstogether with ViraPower™ Lentiviral Expression systems from Invitrogen.

Examples of use of lentiviral vectors for gene therapy for inheriteddisorders and various types of cancer, and these references are herebyincorporated by reference (Klein, C. and Baum, C. (2004). Hematol. J.,5, 103-111; Zufferey, R et. al. (1997). Nat. Biotechnol., 15, 871-875;Morizono, K. et. al. (2005). Nat. Med., 11, 346-352; Di Domenico, C. et.al. (2005), Hum. Gene Ther., 16, 81-90; Kim, E. Y., et. al., (2004).Biochem. Biophys. Res. Comm., 318, 381-390).

Non-retroviral vectors also have been used in genetic therapy. One suchalternative is the adenovirus (Rosenfeld, M. A., et al., Cell 68:143155(1992); Jaffe, H. A. et al., Nature Genetics 1:372-378 (1992);Lemarchand, P. et al., Proc. Natl. Acad. Sci. USA 89:6482-6486 (1992)).Major advantages of adenovirus vectors are their potential to carrylarge segments of DNA (36 Kb genome), a very high titre (10¹¹/ml),ability to infect non-replicating cells, and suitability for infectingtissues in situ, especially in the lung. The most striking use of thisvector so far is to deliver a human cystic fibrosis transmembraneconductance regulator (CFTR) gene by intratracheal instillation toairway epithelium in cotton rats (Rosenfeld, M. A., et al., Cell63:143-155 (1992)). Similarly, herpes viruses may also prove valuablefor human gene therapy (Wolfe, J. H. et al., Nature Genetics 1:379-384(1992)). Of course, any other suitable viral vector may be used forgenetic therapy with the present invention.

U.S. Pat. No. 6,531,456 provides methods for the successful transfer ofa gene into a solid tumor cell using recombinant AAV virions. Generally,the method described in U.S. Pat. No. 6,531,456 allows for the direct,in vivo injection of recombinant AAV virions into tumor cell masses,e.g., by intra-tumoral injection. The invention also provides for thesimultaneous delivery of a second gene using the recombinant AAVvirions, wherein the second gene is capable of providing an ancillarytherapeutic effect when expressed within the transduced cell. U.S. Pat.No. 6,531,456 is hereby incorporated by reference in its entirety.

The viron used for gene therapy can be any viron known in the artincluding but not limited to those derived from adenovirus,adeno-associated virus (AAV), retrovirus, and lentivirus. Recombinantviruses provide a versatile system for gene expression studies andtherapeutic applications.

The recombinant AAV virions described above, including the DNA ofinterest, can be produced using standard methodology, known to those ofskill in the art. The methods generally involve the steps of (1)introducing an AAV vector into a host cell; (2) introducing an AAVhelper construct into the host cell, where the helper construct includesAAV coding regions capable of being expressed in the host cell tocomplement AAV helper functions missing from the AAV vector; (3)introducing one or more helper viruses and/or accessory function vectorsinto the host cell, wherein the helper virus and/or accessory functionvectors provide accessory functions capable of supporting efficientrecombinant AAV (“rAAV”) virion production in the host cell; and (4)culturing the host cell to produce rAAV virions. The AAV vector, AAVhelper construct and the helper virus or accessory function vector(s)can be introduced into the host cell either simultaneously or serially,using standard transfection techniques. Using rAAV vectors, genes can bedelivered into a wide range of host cells including many different humanand non-human cell lines or tissues. Because AAV is non-pathogenic anddoes not illicit an immune response, a multitude of pre-clinical studieshave reported excellent safety profiles. rAAVs are capable oftransducing a broad range of cell types and transduction is notdependent on active host cell division. High titers, >10⁸ viralparticle/ml, are easily obtained in the supernatant and 10¹¹-10¹² viralparticle/ml with further concentration. The transgene is integrated intothe host genome so expression is long term and stable.

A simplified system for generating recombinant adenoviruses is presentedby He T C. et. al. Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998. Thegene of interest is first cloned into a shuttle vector, e.g.pAdTrack-CMV. The resultant plasmid is linearized by digesting withrestriction endonuclease Pme I, and subsequently cotransformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid, e.g. pAdEasy-1of Stratagene's AdEasy™ Adenoviral Vector System. Recombinant adenovirusvectors are selected for kanamycin resistance, and recombinationconfirmed by restriction endonuclease analyses. Finally, the linearizedrecombinant plasmid is transfected into adenovirus packaging cell lines,for example HEK 293 cells (E1-transformed human embryonic kidney cells)or 911 (E1-transformed human embryonic retinal cells) (Human GeneTherapy 7:215-222, 1996). Recombinant adenovirus are generated withinthe HEK 293 cells.

The use of alternative AAV serotypes other than AAV-2 (Davidson et al(2000), Proc. Natl. Acad. Sci. USA 97(7)3428-32; Passini et al (2003),J. Virol. 77(12):7034-40) has demonstrated different cell tropisms andincreased transduction capabilities. With respect to brain cancers, thedevelopment of novel injection techniques into the brain, specificallyconvection enhanced delivery (CED; Bobo et al (1994), Proc. Natl. Acad.Sci. USA 91(6):2076-80; Nguyen et al (2001), Neuroreport 12(9):1961-4),has significantly enhanced the ability to transduce large areas of thebrain with an AAV vector.

Large scale preparation of AAV vectors is made by a three-plasmidcotransfection of a packaging cell line: AAV vector carrying a DNAcoding sequence for an antisense oligonucleotide to hnRNPLL or an siRNAhnRNPLL nucleic acid molecule, AAV RC vector containing AAV rep and capgenes, and adenovirus helper plasmid pDF6, into 50×150 mm plates ofsubconfluent 293 cells. Cells are harvested three days aftertransfection, and viruses are released by three freeze-thaw cycles or bysonication.

AAV vectors are then purified by two different methods depending on theserotype of the vector. AAV2 vector is purified by the single-stepgravity-flow column purification method based on its affinity forheparin (Auricchio, A., et. al., 2001, Human Gene therapy 12; 71-6;Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford,C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5vectors are currently purified by three sequential CsCl gradients.

Pharmaceutical compositions used in the methods described herein can bedelivered systemically via in vivo gene therapy. A variety of methodshave been developed to accomplish in vivo transformation includingmechanical means (e.g, direct injection of nucleic acid into targetcells or particle bombardment), recombinant viruses, liposomes, andreceptor-mediated endocytosis (RME) (for reviews, see Chang et al. 1994Gastroenterol. 106:1076-84; Morsy et al. 1993 JAMA 270:2338-45; andLedley 1992 J. Pediatr. Gastroenterol. Nutr. 14:328-37).

Another gene transfer method for use in humans is the transfer ofplasmid DNA in liposomes directly to human cells in situ (Nabel, E. G.,et al., Science 249:1285-1288 (1990)). Plasmid DNA should be easy tocertify for use in human gene therapy because, unlike retroviralvectors, it can be purified to homogeneity. In addition toliposome-mediated DNA transfer, several other physical DNA transfermethods, such as those targeting the DNA to receptors on cells byconjugating the plasmid DNA to proteins, have shown promise in humangene therapy (Wu, G. Y., et al., J. Biol. Chem. 266:14338-14342 (1991);Curiel, D. T., et al., Proc. Natl. Acad. Sci. USA, 88:8850-8854 (1991)).

For gene therapy viruses, the dosage ranges from 10⁶ to 10¹⁴ particlesper application. Alternatively the biolistic gene gun method of deliverymay be used. The gene gun is a device for injecting cells with geneticinformation, originally designed for plant transformation. The payloadis an elemental particle of a heavy metal coated with plasmid DNA. Thistechnique is often simply referred to as biolistics. Another instrumentthat uses biolistics technology is the PDS-1000/He particle deliverysystem. The proteins, expression vector, and/or gene therapy virus canbe coated on minute gold particles, and these coated particles are“shot” into biological tissues such as hemangiomas and melanoma underhigh pressure. An example of the gene gun-based method is described forDNA based vaccination of cattle by Loehr B. I. et. al. J. Virol. 2000,74:6077-86.

The present invention may be defined by any of the followingalphabetized paragraghs:

-   -   [A] A method of diagnosing membranous nephropathy (MN) in a        subject, the method comprising detecting the presence of        antibodies that are reactive to a phospholipase A2 receptor        (PLA2R), wherein the antibodies are found in a sample from a        subject.    -   [B] The method of paragragh [A], wherein the MN is idiopathic.    -   [C] The method of paragragh [A], wherein the subject is a human.    -   [D] The method of any of paragraghs [A]-[C], wherein a kidney        biopsy is not performed.    -   [E] The method of paragragh [A], wherein the PLA2R is a        mammalian PLA2R.    -   [F] The method of paragragh [A], wherein the sample is a blood        sample.    -   [G] The method of paragragh [A], wherein the antibodies are of        the IgG subclass: IgG1-4.    -   [H] The method of any of paragraghs [A]-[G], wherein the        detecting is performed by a serological immunoassay.    -   [I] A method of prognosis evaluation in a subject being treated        for MN, the method comprising:        -   a. determining at a first time point a level of antibodies            that are reactive to a PLA2R, wherein the antibodies are            found in a sample from a subject;        -   b. determining at a second time point a level of antibodies            that are reactive to a PLA2R, wherein the second time point            is after the first time point; and        -   c. comparing the levels of antibodies of the two time            points, wherein a decrease in the level of antibodies in the            second time point compared to the first time point indicates            that the treatment is effective.    -   [J] A method of prognosis evaluation in a subject for MN, the        method comprising:        -   a. determining at a first time point a level of antibodies            that are reactive to a PLA2R, wherein the antibodies are            found in a sample from a subject; and        -   b. determining at a second time point a level of antibodies            that is reactive to a PLA2R, wherein the second time point            are after the first time point;        -   wherein when the level of antibodies in the second time            point decreases to below a detection limit indicates that            there is remission.    -   [K] A method of prognosis evaluation in a subject for MN, the        method comprising:        -   a. determining at a first time point a level of antibodies            that are reactive to a PLA2R, wherein the antibodies are            found in a sample from a subject;        -   b. determining at a second time point a level of antibodies            that are reactive to a PLA2R, wherein the second time point            is after the first time point;        -   c. comparing the levels of antibodies of the two time            points, wherein an increase in the level of antibodies in            the second time point compared to the first time point            indicates that there is relapse of membranous nephropathy.    -   [L] The method of paragraghs [I], [J] or [K], wherein the MN is        idiopathic.    -   [M] The method of paragraghs [I], [J] or [K], wherein the        subject is a human.    -   [N] The method of paragraghs [I], [J] or [K], wherein a kidney        biopsy is not performed.    -   [O] The method of paragragh [I], [J] or [K], wherein the PLA2R        is a mammalian PLA2R.    -   [P] The method of paragragh [I], [J] or [K], wherein the sample        is a blood sample.    -   [Q] The method of paragragh [I], [J] or [K], wherein the        antibodies are of the IgG subclass: IgG1-4.    -   [R] The method of any of paragraghs [L]-[Q], wherein the        detecting is performed by a serological immunoassay.    -   [S] The method of paragragh [I], wherein the treatment is an        immunosuppressive treatment.    -   [T] A method of treatment of MN in a subject, the method        comprising removing an antibody that is reactive to a PLA2R from        a sample in a subject ex vivo.    -   [U] The method of paragragh [T], wherein the subject is a human.    -   [V] The method of paragragh [T], wherein the MN is idiopathic.    -   [W] The method of paragragh [T], wherein the phospholipase A2        receptor is a mammalian PLA2R.    -   [X] The method of paragragh [T], wherein the sample is a blood        sample.    -   [Y] The method of paragragh [T], wherein the antibodies are of        the IgG subclass: IgG 1-4.    -   [Z] The method of paragragh [T], wherein the antibodies are        removed from the blood by immunoabsorption.    -   [AA] The method of paragragh [T], wherein the sample is returned        back into the subject after the removal of the antibodies.    -   [BB] A method of treatment of MN in a subject, the method        comprising administering an effective amount of PLA2R or        fragments thereof or a vector expressing a PLA2R or fragments        thereof.    -   [CC] The method of paragragh [BB], wherein the MN is idiopathic.    -   [DD] The method of paragragh [BB], wherein the subject has        tested positive for antibodies reactive against a PLA2R.    -   [EE] The method of paragragh [BB], wherein the phospholipase A2        receptor is a mammalian PLA2R.    -   [FF] A composition for the treatment of idiopathic MN, the        composition comprising a PLA2R or fragments thereof.    -   [GG] A use of an effective amount of PLA2R or fragments thereof        or a vector expressing a PLA2R or fragments thereof for the        treatment of MN in a subject.    -   [HH] A use of an effective amount of PLA2R or fragments thereof        or a vector expressing a PLA2R or fragments thereof in the        manufacture of a medicament for treatment of MN in a subject.    -   [II] The use of paragragh [GG] or [HH], wherein the MN is        idiopathic.    -   [JJ] The use of paragragh [GG] or [HH], wherein the subject has        tested positive for antibodies reactive against a PLA2R    -   [KK] The use of paragragh [GG] or [HH], wherein the        phospholipase A2 receptor is a mammalian PLA2R.    -   [LL] An immunoassay comprising:        -   a. contacting a sample from a subject with a PLA2R or PLA2R            fragment thereof;        -   b. forming an antibody-protein complex between the antibody            present in a sample with the PLA2R or PLA2R fragment            thereof;        -   c. washing to remove any unbound antibody;        -   d. adding a detection antibody that is labeled and is            reactive to the antibody from the sample;        -   e. washing to remove any unbound labeled detection antibody;            and        -   f. converting the label to a detectable signal, wherein the            presence of a detectable signal indicates the likelihood of            MN in the subject.    -   [MM] The immunoassay of paragragh [LL], wherein the MN is        idiopathic.    -   [NN] The immunoassay of paragragh [LL] or [MM], wherein, the        subject is a human.    -   [OO] The immunoassay of paragragh [LL], [MM] or [NN], wherein        the sample is a blood sample.    -   [PP] The immunoassay of any of paragragh [LL]-[OO], wherein a        kidney biopsy is not performed in the subject.    -   [QQ] The immunoassay of any of paragragh [LL]-[PP], wherein the        PLA2R is a mammalian PLA2R.    -   [RR] The immunoassay of any of paragragh [LL]-[QQ], wherein the        antibodies are of the IgG subclass: IgG1-4.    -   [SS] The immunoassay of any of paragragh [LL]-[RR], wherein the        PLA2R or PLA2R protein fragment thereof is deposited or        immobilized on a solid support.    -   [TT] The immunoassay of any of paragragh [LL]-[SS], wherein a        known amount of a PLA2R or PLA2R protein fragment is deposited        or coupled to a solid support.    -   [UU] The immunoassay of paragragh [SS] or [TT], wherein the        support is in the format of a dipstick, a test strip, a latex        bead, a microsphere or a multi-well plate.    -   [VV] The immunoassay of any of paragragh [LL]-[UU], wherein the        detection antibody is labeled by covalently linking to an        enzyme, label with a fluorescent compound or metal, or label        with a chemiluminescent compound.    -   [WW] The immunoassay of any of paragragh [LL]-[VV], wherein the        detection antibody is specifically reactive to the subject.    -   [XX] The immunoassay of any of paragragh [LL]-[WW], wherein the        detectable signal is compared to a set of detectable signals        from a titration curve derived from immunoassays of known        amounts of PLA2R or fragments in increasing quantity.    -   [YY] The immunoassay of any of paragragh [LL]-[XX], wherein the        immunoassay is performed for a plurality of samples from a        subject obtained over a period of time.    -   [ZZ] The immunoassay of paragragh [YY], wherein the plurality of        samples is obtained every two or three months for at least a two        year period.    -   [AAA] The immunoassay of paragragh [ZZ], wherein the detectable        signal of each immunoassay is compared to the detectable signal        of a sample obtained from a consecutively prior time point,        wherein a reduction of 10% of detectable signal indicates        effective treatment of MN in the subject.    -   [BBB] An immunoassay comprising:        -   a. contacting a sample from a subject with a PLA2R or PLA2R            fragment thereof;        -   b. forming an antibody-protein complex between the antibody            present in a sample with the PLA2R or PLA2R fragment            thereof;        -   c. measuring a light scattering intensity resulting from the            formation of the antibody-protein complex wherein the light            scattering intensity of at least 10% above a control light            scattering intensity indicates the likelihood of MN or            relapse of MN in the subject.    -   [CCC] The immunoassay of paragragh [BBB], wherein the PLA2R or        PLA2R protein fragment thereof is immobilized on a solid        support.    -   [DDD] The immunoassay of paragragh [CCC], wherein the solid        support is a latex bead or a microsphere.    -   [EEE] The immunoassay of any of paragragh [BBB]-[DDD], wherein        the control light scattering intensity is that of PLA2R or PLA2R        protein fragment in the absence of sample.    -   [FFF] The immunoassay of any of paragragh [BBB]-[EEE], wherein        light scattering intensity is measured in a nephelometer.    -   [GGG] The immunoassay of any of paragragh [BBB]-[FFF], wherein        the immunoassay is performed for a plurality of samples from a        subject obtained over a period of time.    -   [HHH] The immunoassay of paragragh [GGG], wherein the plurality        of samples are obtained every two or three months for at least a        two year period.    -   [III] The immunoassay of paragragh [HHH], wherein the light        scattering intensity of each immunoassay is compared to the        light scattering intensity of a sample obtained from a        consecutively prior time point, wherein a reduction of 10% of        light scattering intensity indicates effective treatment of MN        in the subject.    -   [JJJ] A device for identifying the presence or the level of        antibodies that are reactive to a PLA2R in a sample from a        subject comprising:        -   a. at least a PLA2R protein or fragments thereof; and        -   b. at least one solid support wherein the PLA2R protein or            fragments thereof is deposited on the support.    -   [KKK] The device of paragragh [JJJ], wherein at least a PLA2R        protein or fragments thereof that is deposited on the solid        support is immobilized on the support.    -   [LLL] The device of paragragh [JJJ], wherein the solid support        is in the format of a dipstick, a test strip, a latex bead, a        microsphere or a multi-well plate.    -   [MMM] The device of paragragh [JJJ], wherein the subject is a        human.    -   [NNN] The device of paragragh [JJJ], wherein a kidney biopsy is        not performed in the subject.    -   [OOO] The device of paragragh [JJJ], wherein the sample from the        subject is a blood sample.    -   [PPP] The device of paragragh [JJJ], wherein the PLA2R protein        is a human or pig PLA2R protein.    -   [QQQ] The device of paragragh [JJJ] further comprising a second        labeled PLA2R protein or fragments thereof which produces a        detectable signal.    -   [RRR] The device of paragragh [JJJ] further comprising a        detection antibody, wherein the detection antibody is specific        for the antibodies that are reactive to a PLA2R in the sample of        the subject and the detection antibody produces a detectable        signal.    -   [SSS] The device of paragragh [JJJ], wherein the device performs        an immunoassay wherein an antibody-protein complex is formed.    -   [TTT] The device of paragragh [SSS], wherein the immunoassay is        a serological immunoassay.    -   [UUU] The device of paragragh [SSS], wherein the immunoassay is        a nephrelometric immunoassay    -   [VVV] The use of any of the devices of paragraghs [JJJ]-[SSS]        for facilitating the diagnosis of membranous nephropathy in a        subject, wherein a detectable amount of antibodies that are        reactive to a PLA2R indicates likelihood of membranous        nephropathy in the subject.    -   [WWW] A kit comprising a device of paragragh [JJJ] and a        detection antibody, wherein the detection antibody is specific        for the antibodies that are reactive to a PLA2R in the sample of        the subject and produces a detectable signal.    -   [XXX] A kit comprising a device of paragragh [JJJ] and a second        labeled PLA2R protein or fragments thereof that produces a        detectable signal.    -   [YYY] A kit comprising a device of paragragh [JJJ] and a        nephelometer cuvette.    -   [ZZZ] A system comprising:        -   a. a measuring module measuring auto-antibody information            comprising a detectable signal from an immunoassay            indicating the presence or level of antibodies that are            reactive to a PLA2R from a sample obtained form a subject;        -   b. a storage module configured to store data output from the            measuring module;        -   c. a comparison module adapted to compare the data stored on            the storage module with reference and/or control data, and            to provide a retrieved content, and        -   d. an output module for displaying the retrieved content for            the user, wherein the retrieved content the presence of            detectable amount of antibodies reactive against PLA2R            indicates that the subject has MN or has a relapse of MN.    -   [AAAA] The system of paragragh [ZZZ], wherein the control data        comprises data from a population of non-MN healthy individuals.    -   [BBBB] A system to facilitate the prognosis evaluation of MN in        a subject, comprising:        -   a. a determination module configured to receive and output            auto-antibody information to a PLA2R from a sample obtained            from a subject, wherein the auto-antibodies information            measures the level of auto antibodies that are reactive to            the PLA2R;        -   b. a storage module configured to store output information            from the determination module;        -   c. a comparison module adapted to compare the data stored on            the storage module with reference and/or control data, and            to provide a comparison content, and        -   d. an output module for displaying the comparison content            for the user, wherein if there is no detectable amount of            auto antibodies reactive against PLA2R then the subject is            in remission or if there is a reduction of at least 10% to a            prior reading, then the treatment for MN is effective in the            subject.    -   [CCCC] The computer system of paragragh [BBBB], wherein the        control data comprises previous data from the same subject        wherein the previous data had indicated detectable amounts of        auto-antibodies.    -   [DDDD] A computer readable storage medium comprising:        -   a. a storing data module containing data from a sample            obtained from a subject that represents a signal level from            an immunoassay for antibodies that are reactive to a PLA2R;        -   b. a comparison module that compares the data stored on the            storing data module with a reference data and/or control            data, and to provide a comparison content, and        -   c. an output module displaying the comparison content for            the user, wherein the presence of a detectable amount of            antibodies reactive against PLA2R of at least 10% relative            to the reference data and/or control data indicates that the            subject has MN or has a relapse of MN.    -   [EEEE] The system of paragragh [DDDD], wherein the control data        comprises data from a population of non-MN healthy individuals.

This invention is further illustrated by the following example whichshould not be construed as limiting. The contents of all referencescited throughout this application, as well as the figures and table areincorporated herein by reference.

Example Materials and Methods Human Sera

With approval from the Institutional Review Board at Boston University,we have collected and stored coded serum samples from patients withmembranous nephropathy, other glomerular or autoimmune disorders, andnormal volunteers. Those classified as having idiopathic MN have hadbiopsy-proved MN in the absence of traditional secondary features, suchas positive anti-nuclear antibodies (ANA), anti-double-stranded DNAantibodies, or hepatitis B serologies. Further classification into thesegroups is discussed in the Supplemental Information.

Human Kidney Tissue

We obtained human kidneys that were unsuitable for transplantation anddonated for research from the New England Organ Bank. Glomeruli werecollected from minced kidney cortex by filtering through metal sieves(ref) and were resuspended and extracted in a detergent-containing RIPAbuffer (Boston BioProducts, Boston, Mass.). Contaminating IgG wasremoved from this preparation via incubation with Immobilized Protein GPlus (Thermo Fisher). We used peptide N-glycosidase F (PNGase F; NewEngland Biolabs) in the absence of reducing agent to remove N-linkedsugar residues from the glomerular proteins when indicated. In order topartially purify the glomerular glycoproteins, we passed humanglomerular extract over wheat germ agglutinin (WGA) agarose bead column(Vector Laboratories) and eluted the bound glycoproteins with 500 mMN-acetyl glucosamine (GlcNAc). Both native and the 200 kDa PNGaseF-deglycosylated antigen were found to bind the column.

Western Blot Protocol

Human glomerular extract or cell-expressed human PLA2R waselectrophoresed under non-reducing conditions and transferred tonitrocellulose membranes according to standard protocols. Weimmunoblotted with human serum as the primary antibody, typically at1:100 to 1:250, and horseradish peroxidase-conjugated donkey anti-humanIgG secondary antibody (Jackson ImmunoResearch) at 1:40,000. The PLA2Rantibody used for these experiments is a polyclonal guinea pig antibodyraised against the full-length purified rabbit PLA2 receptor. Itrecognizes the human protein under both reducing and non-reducing gelelectrophoresis conditions (Granata, F., et al. 1995, J. Immunol. 174:464-74; G. Lambeau, personal communication). We purchased sheepantibodies against the four IgG subclasses from The Binding Site andused them at the dilutions recommended by the manufacturer.

Mass Spectrometry Analysis and Data Interpretation

We excised gel regions of interest and performed in-gel trypsindigestion as previously described in Powell, 2003 Mol Cell Biol23:5376-5387. We analyzed the resulting peptides with a modified versionof a previously described method that couples liquid chromatography (LC)with tandem mass spectrometry (MS/MS) (Powell, 2004, Mol Cell Biol24:7249-7259). We used the acquired MS data to search the NCBI RefSeqHuman database using the SEQUEST algorithm and analyzed the data withSequestSorcerer™ (Sage-N Research, San Jose, Calif.). The enrichment orrelative abundance of each identified protein was determined bynormalizing the number of spectral counts matching to the protein by itspredicted molecular weight. This value has been termed a ProteinAbundance Factor (PAF) (Powell, 2004, Mol Cell Biol 24:7249-7259).

Immunohistology

We froze fresh sections of human kidney in Optimal Cutting Temperaturesolution (TissueTek) and cut 4 micron sections with a cryotome. Weobtained serial frozen sections from five MN kidney biopsies from Dr.Helmut Rennke (Boston, Mass.). We fixed and permeabilized the sectionswith methanol:acetone and blocked with 10% bovine serum albumin in TBS.To detect PLA2R, we used guinea-pig anti-rabbit PLA2R at 1:400 andCy3-conjugated donkey anti-guinea pig IgG (Jackson ImmunoResearch) at1:500. To demonstrate specificity of the staining, we precleared thepolyclonal antibody with a fragment of rabbit PLA2R containing the 4thto 6th lectin-binding domains. This significantly depleted theimmunofluorescence signal to PLA2R.

The diagnosis of membranous nephropathy was established by renal biopsyin all cases. Those classified as idiopathic MN had no evidence ofsecondary features, which include positivity for anti-nuclear antibodiesor anti-double stranded DNA, hepatitis B antigenemia, or electron-densedeposits on renal biopsy in locations other than subepithelial. We didnot make an attempt to rule-out occult malignancy as a potential causeof secondary MN. The other glomerular disorders were diagnosed by biopsy(2 FSGS; 1 DN; 1 Henoch-Shonlein purpura) or by clinical features. Theseincluded longstanding MN with gradually-progressive proteinuria,orthostatic proteinuria evidenced by split urine collections. Thepatients with additional autoimmune or rheumatologic conditions includedsystemic lupus erythematosus without significant proteinuria,dermatomyositis, scleroderma/mixed connective tissue overlap disease,and bullous pemphigus.

Given the size similarity to IgG, we initially excluded IgG as thetarget of what could have been a rheumatoid factor-like activity in themembranous serum. Glomerular extracts were treated with protein G-linkedagarose beads to remove contaminating IgG that was invariably present inthe glomerular extract. Conversely, MN sera were incubated withheat-aggregated IgG covalently linked to Affi-Gel 10 beads to pre-adsorbout any serum factors that were reactive with IgG. Serum samples treatedin his manner demonstrated an identical reactivity with the 200 kDaantigen as did the starting serum (data not shown). Additionally, wewere able to show subtle differences in migration between IgG and theMN-Ag on low percentage (6%) agarose gels run for extended periods oftime, and did not note a major shift in the size of IgG when treatedwith PNGase F. Despite our confidence that the target antigen was not animmunoglobulin, the size similarity to human IgG and the necessity ofrunning the gels under non-reducing conditions in order to detected theantigen made immunoprecipitation of the MN-Ag nearly impossible, despitevaried approaches. We therefore approached the task of identifying thisputative target antigen in membranous nephropathy (MN-Ag) usingbiochemical purification techniques.

Results

MN Sera React with a 200 KDA Glomerular Protein

Our approach to the identification of the target antigen in humanmembranous nephropathy was based on the presumption that autoantibodiesin the serum of patients with MN would identify candidate bands bystandard western blotting of human glomerular proteins. Aconsistently-identified band was not detected until we fortuitouslyelectrophoresed the proteins under non-reducing conditions, when aprominent, approximately 200 kDa band was detected by several of serathat had previously been negative using the more-standard reducingconditions. Testing of other banked and newly-collected sera frompatients with idiopathic MN showed similar reactivity in over 50% ofsuch patients. In FIG. 1A, the five MN sera all recognize a band ofapproximately 200 kDa, whereas the sera from the nephrotic controlpatients do not. In the lower panel of FIG. 1A, these five reactive MNsera are used to western blot alternating lanes of native anddeglycosylated (PNGase F+) glomerular proteins. All five MN sera reactidentically with the 200 kDa native antigen and an approximately 150 kDadeglycosylated protein. Strikingly, serum from normal volunteers (n=23),patients with other nephrotic conditions such as diabetic nephropathyFSGS (n=13), or patients with other autoimmune, rheumatologic disorders(n=6) were all non-reactive toward this antigen when assayed underidentical conditions. Further analysis of the patients with MN revealedthat none of the eight cases of secondary MN (6 lupus-associated and 2hepatitis B-associated) were reactive for the 200 kDa antigen. Removalof N-linked carbohydrate chains with peptide N-glycosidase (PNGase) Fcaused a significant shift in mobility of this antigen to approximately150 kDa, indicating that it is heavily glycosylated. All sera initiallyreactive with the native 200 kDa band also identified the smaller,deglycosylated band (FIG. 1A).

To verify the specificity of anti-PLA2Rautoantibodies positivereactivity for idiopathic membranous nephropathy, we have increased thenumber of samples from subjects tested for antibodies to PLA2R bywestern blotting. We also increased the controls to n=32 and patientswith other nephrotic conditions such as diabetic nephropathy FSGS(n=25). In addition to an increase in the number of subjects withidiopathic MN in Boston (MA, USA), we have received and tested samplesfrom the Mayo Clinic in Rochester, (MN, USA), the University of Lund inSweden, and the University in the Netherlands. We have also increasedthe number of control samples from patients with secondary MN, otherautoimmune and kidney diseases, as disease controls and normal subjects.Approximately 72-82% of patients with idiopathic MN tested positive foranti-PLA2R antibodies whereas none of the normal or disease controls, orpatients with secondary forms of MN was positive (FIG. 1C).

Given the evidence of significant glycosylation, we tested the abilityof the antigen to bind various lectin columns, and found that it boundto wheat-germ agglutinin (WGA) in both its native and N-deglycosylatedform. The binding of both forms may reflect residual N-linkedcarbohydrates that are inaccessible to PNGaseF under the non-reducingconditions required to maintain antigenicity. Native and deglycosylatedhuman glomerular proteins were eluted from WGA and electrophoresed; thetwo gel regions corresponding to the 200 and 150 kDa antigen bands on WBwere excised, subjected to in-gel tryptic digestion and analyzed by massspectrometry. Under the assumption that peptide sequences correspondingto the putative target antigen would be identified in both samples, wegenerated a list of candidate identities for the putative MN antigen(see Table 1). Using available antibodies and/or recombinant proteins,we assessed these candidate antigens and were fortunate to encounter apossible match when antibodies to the M-type phospholipase A2 receptoridentified a similarly-sized protein in human glomeruli (FIG. 2A).

Phospholipase A2 Receptor in the Target Antigen in MN

Both MN serum and the anti-PLA2R recognize identical bands in WB(Westren blots) of glomeruli extracts (FIG. 2A). The recombinant proteinmigrates to a slightly lower position than the native glomerularprotein, although deglycosylation with PNGase F causes both to migrateto the same position. Cell-expressed recombinant human PLA2R (rPLA2R)blotted with MN sera yielded a distinct band on WB that was slightlysmaller than the corresponding signal from human glomeruli. However,when both samples were deglycosylated, they migrated to the sameposition (FIG. 2A). This suggests a small difference in overallglycosylation between the native protein and the recombinant form.Importantly, WB of these same samples with a monospecific polyclonalantibody against PLA2R revealed an identical pattern. We have sinceconfirmed that all MN samples that reactive with the 200 kDaglycoprotein from human glomeruli also recognize rPLA2R, confirming thatthe 200 kDa band from human glomeruli that we had been investigating wasindeed PLA2R.

Human sera that are reactive with the 200 kDa MN-Ag by WB canimmunoprecipitate (IP) PLA2R from human glomerular extracts (FIG. 2B).All three reactive MN sera are able to IP PLA2R from both human and pigglomerular protein extracts, whereas the controls do not. Appreciableamounts of starting IgG were present in all cases; the amount is lowerin lane 2 as this patient was particularly nephrotic. Two of the threenon-reactive sera and all three nephrotic control sera did not IP PLA2Runder identical conditions. Interestingly, with one of the serainitially found to be non-reactive by WB, a faint band was detected byIP (not visible in the reproduction). When the serum was re-assayed at a1:25 dilution, it was found to identify rPLA2R by WB (data not shown).This may suggest that other MN patients initially thought not to haveanti-PLA2R autoantibodies may instead have low-level titers not easilydetectable by our initial WB assay (screening is typically done at a1:100 serum dilution).

In addition, FIG. 2C shows that the glomerular glycoprotein identifiedby reactive MN sera is the human phospholipase A2 receptor. Whole humanserum was used to immunoprecipitate (IP) glomerular proteins, and theIP's were then electrophoresed and Western blotted with an antibodyspecific to the M-type phospholipase A2 receptor. The first five lanesshow IP's with sera that were known to be positive by WB (as in FIG. 1).The 6th lane represents an IP with serum from a patient with MN that wasknown to be negative. Lanes 7 and 8 show IP's with serum from normalvolunteers, and in the final lane, human serum was omitted from the IPto rule out non-specific binding of glomerular proteins to the agarosebeads.

Recombinant PLA2R shares the reduction-sensitive epitope as does thenative glomerular protein (FIG. 3A). In the WB in which equal amounts ofhuman glomerular extract (HGE) were electrophoresed under reducing andnon-reducing conditions; recombinant PLA2R was treated similarly. WB wasperformed with reactive MN serum or a polyclonal anti-PLA2R antibody anddetected with appropriate secondary antibodies. The reactivity towardsthe native or recombinant PLA2R is in the non-reduced state, for both MNserum and the polyclonal anti-PLA2R were noted. However, whileanti-PLA2R recognizes the antigen in reduced form, MN serum fails todetect the reduced native or recombinant protein. Both the monospecificanti-PLA2R antiserum and MN sera strongly detect recombinant and nativeglomerular PLA2R by WB under non-reducing conditions. Whereas thepolyclonal antibody still detects both forms (albeit less robustly) whenrun under reducing conditions, MN sera fail to react with either form.

The IgG classes of the auto antibodies reactive against PLA2R, humanglomerular proteins, recombinant PLA2R, and antigens from E. coli wereblotted with a single reactive MN serum, followed by antibodies specificfor the IgG subclasses. The predominant subclass that reacts with nativeor PLA2R is IgG4, whereas it is IgG2 for a 70 kDa E. coli proteinincluded as a control. The relative amounts of IgG subclasses in thisparticular serum were assessed by WB of diluted total serum with thesubclass-specific antibodies. The IgG2 form is not well-detected in itsdenatured form, but is clearly present as detected by its binding to the70 kDa E. coli protein. It is well established that the IgG antibodiesthat are detected by immunofluorescence microscopy in the glomeruli ofpatients with idiopathic MN are of the IgG4 subclass. Here we found thatthe IgG antibodies in the serum of patients with idiopathic MN thatreacted with PLA2R were also of the IgG4 subclass. We have furtherconfirm this observations inadditional serum from 6 patients diagnosedwith idiopathic MN. Human glomerular extract (HE) was blotted initiallywith serum samples from six patients with idiopathic MN (MN1 throughMN6), followed by sheep antibodies specific for each human IgG subclass(1 through 4), and was detected with peroxidase conjugated antisheep IgGantibody. The predominant IgG subclass that reacted with the nativeantigen was IgG4, with varying amounts of reactivity seen for IgG1,IgG2, and IgG3. Identical results were obtained with the use ofrecombinant PLA2R instead of HGE (data not shown). The immunoglobulinresponse in membranous nephropathy is typical of a Th2 response, with apredominance of IgG4 subclass (Kuroki, 2005, Kidney Int 68:302-310).When either human glomerular proteins or rPLA2R are immunoblotted withappropriately-reactive MN patient serum, the predominant subclassdetected by WB is IgG4 (FIG. 3B), whereas it is IgG2 for an unrelatedbacterial antigen.

Glomerular Location of the Phospholipase A2 Receptor

The proposed pathomechanism for human MN is that autoantibodies bind insitu to an antigen present on the podocyte. Because PLA2R has beendescribed in a soluble form, we first discounted the possibility thatcirculating antibody-PLA2R complexes were being trapped in the GBM.Neither MN sera nor control sera had any detectable PLA2R, even afterenrichment by means of lectin binding (data not shown). Nor could wedetect circulating immune complexes of PLA2R-IgG through eitherprecipitation with polyethylene glycol 6000 or protein Gimmunoprecipitation of IgG in serum samples from either group.Conversely, we were able to detect the presence of PLA2R on podocytes byimmunofluorescence with the monospecific anti-PLA2R antibody. Frozensections of normal human kidney cortex were co-stained with anti-agrinfollowed by a FITC-conjugated anti-rabbit secondary antibody, to labelthe glomerular basement membrane (GBM) and anti-PLA2R followed byCY3-conjugated anti-guinea pig secondary antibody (data not shown). Wefound that the PLA2R signal is clearly present external to the GBM, andlocalizes to both the cell body and processes of the podocyte. Thisstaining is markedly reduced when the antibody is precleared with arecombinant fragment of PLA2R containing CRD domains 4-6 (data notshown). The staining pattern is granular, and extends from the cell bodyto the basal foot processes. When cryosections are stained by dualimmunofluorescence with antibodies against PLA2R and agrin, a componentof the glomerular basement membrane (GBM), the podocyte signal isclearly seen immediately adjacent and external to the GBM. The majorityof glomerular and podocyte PLA2R staining can be blocked bypreincubation of the antibodies with recombinant PLA2R fragmentscontaining CTLDs 4, 5. and 6.

Next we studied the localization of the anti-PLA2R IgG4 in glomerulus.The PLA2R is present in a granular pattern in membranous nephropathybiopsy specimens, and colocalizes with IgG4. A frozen section of abiopsy specimen from a patient diagnosed with MN reveals PLA2R and IgG4that colocalize well in the peripheral capillary walls and GBM (data notshown). A serial section of the same patient is stained in the samemanner, although the anti-IgG4 antibody was omitted to exclude thepossibility that the anti-sheep secondary antibody was detecting guineapig or donkey IgG, or that bleed through from the Cy3 channel wascausing the signal seen previously. In contrast to podocytes, mesangialcells in normal human kidney tissue did not show staining for PLA2R(data not shown).

Studies in the rat Heymann nephritis model have suggested that “cappingand shedding” of receptor-antibody complexes are depositedsubepithelially into the GBM, and we anticipated that the same is truein the case of PLA2R in human MN. PLA2R is present in a fine granularpattern lining the GBM upon immunofluorescence of cryosections fromkidney biopsy specimens obtained from patients with MN. This could beblocked with the blocking fragment of recombinant PLA2R. Although theintensity of staining varied between the four patient samples weexamined, all revealed the same granular pattern for PLA2R (data notshown). Of interest, PLA2R staining of the podocyte cell body, which hadbeen strong in normal glomerular sections, was greatly attenuated in theMN biopsy samples. Moreover, the GBM staining pattern closely matchedthat of IgG4 on dual immunofluorescence.

Immunofluorescent microscopy showed that anti-PLA2R and IgG4 co-localizein the glomeruli of patients with idiopathic MN but not lupus-associatedMN. To confirm that the IgG in the glomeruli from patients withmembranous nephropathy was reactive with PLA2R, we eluted IgG frombiopsy specimens and used it in Western blotting with native andrecombinant PLA2R. IgG was successfully eluted from four biopsy samplesfrom patients with idiopathic membranous nephropathy, one from patientswith lupus membranous nephropathy, and two from patients with IgAnephropathy. The IgG eluted from the samples from patients withidiopathic membranous nephropathy specifically detected theappropriately sized

PLA2R bands in human glomerular extract and cell lysates that werepositive for recombinant PLA2R (FIGS. 4A and 4B), whereas the IgG elutedfrom the three other samples from patients with immune-complexglomerular disease did not. IgG was eluted from biopsy cores frompatients with idiopathic MN (MN), lupus MN (LMN), or IgA nephropathy(IgAN). This eluted IgG was used to immunoblot human glomerular extract(HGE) or recombinant PLA2R (rPLA2R).

Association with Disease Activity

We have obtained serial serum samples when possible, and have examinedthe change in reactivity in several individuals who have achievedtreatment or spontaneous remission, or alternatively, relapse. Thepresence of autoantibodies to PLA2R, in general, parallels the clinicalsignificant disease activity as measured by urinary protein and serumalbumin (FIGS. 5 and 6). Importantly, a decline or disappearance ofanti-PLA2R antibody can be seen prior to a disappearance of proteinuria.This is understandable given the time required for clearance of immunedeposits and recovery of the podocyte architecture and functional slitdiaphragms.

We have studied additional patients before and after treatment-inducedremission. We found that patients that were positive for anti-PLA2Rbefore treatment became negative after remission was induced withimmunosuppressive treatment (FIGS. 6A and B). The filled squares in thegraph represent urine protein excretion. After treatment was startedwith cyclophosphamide and prednisone, urine protein excretion declinedand reactivity to PLA2R disappeared as shown in the western blot.Similar results have been found in patients treated with rituximab andsynthetic ACTH.

These findings support the utility of using an immunoassay to PLA2R notonly for diagnosis of MN, but also for monitoring disease activityduring treatment or while awaiting a spontaneous remission.

In conclusion, we have identified the M-type phospholipase A2 receptoras the major target antigen in the autoimmune glomerular disease,idiopathic membranous nephropathy. The protein is present on normalhuman podocytes, and over fifty percent of patients with MN bearantibodies reactive with this protein. Furthermore, the protein ispresent within immune deposits in biopsy specimens of patients. Levelsof antibody against PLA2R appear to correlate with disease activity, andmay prove to be a useful method for initial diagnosis of MN and forfollowing disease activity with treatment or while waiting forspontaneous remission.

All references, including any patents or patent applications cited inthis specification, as well as the figures and table, are herebyincorporated by reference. No admission is made that any referenceconstitutes prior art. The discussion of the references states whattheir authors assert, and the applicants reserve the right to challengethe accuracy and pertinence of the cited documents. It will be clearlyunderstood that, although a number of prior art publications arereferred to herein, this reference does not constitute an admission thatany of these documents form part of the common general knowledge in theart, in the United States of America or in any other country.

Example 2

The levels of anti-PLA2R auto-antibodies described herein can also bedetermined using test strips as illustrated in FIG. 8-9. In the teststrip, the membrane is divided into three separate regions: a sample (S)position at one end of the membrane, a test (T) position located at themiddle of the membrane, and a control (C) position found at the oppositeend the membrane (FIG. 8A). Located at S is an excess quantity ofdehydrated PLA2R. The PLA2R can be conjugated to colloidal gold beads orlatex beads for visualization purposes. At T, there is an excessquantity of anti-IgG immobilized on the membrane. At C, there is anotherimmobilized anti-PLA2R antibody (FIG. 8A).

The excess quantity of dehydrated PLA2R at S position is such that whena sample (e.g. serum) is applied at S, anti-PLA2R antibody and PLA2Rcomplexes are formed and free PLA2R are still available to bind theimmobilized anti-PLA2R at position C.

The S position is where a sample of serum is applied. The arrowheadsdelineate the boundary limit that the sample serum should not cross onthe membrane when applying the serum to the membrane. The water in theserum rehydrates the PLA2R. An antibody-protein complex is produced whenthe auto-antibody reactive to PLA2R forms a complex with the rehydratedPLA2R. A mixture of the antibody-protein complexes and non-complexedPLA2R move by capillary action away from position S towards position Tand C.

Upon arrival at the T position, the antibody-protein complex will bindthe immobilized anti-IgG antibody and be immobilized at the T position.The localized concentration of antibody-protein complex that iscolloidal gold or latex bead labeled will become visible as a coloredline at the T position (FIG. 9, middle). The greater the amount ofauto-antibodies in the sample, the broader the visible band at T. Whenthe area remains clear at the T position, this means that there is areanti-PLA2R auto-antibodies (FIG. 9, left). At the C position the freeand labeled PLA2R will be bound and captured by the immobilizedanti-PLA2R antibody. This will in turn result in a concentration of acolloidal gold or latex bead labeled PLA2R at the C position and willbecome visible as colored line at the C position. The C position resultserves as a test control that there is functional PLA2R in the testmaterial and should always be present (FIG. 9, right).

The test strip can be designed in a form of a dipstick test strip (FIG.8B). As a dipstick test strip, the strip is dipped into a sample ofserum at the S position end with sample level not to exceed the boundarylimit. The strip is then laid horizontally with the membrane surfacefacing up on a flat surface. A fixed amount of time is given for theantibody re-hydration, capillary action, and antibody binding reactionto take place. At the end of the fixed time, there should be visiblebands at the C position and depending on the level of auto-anti-PLA2Rantibody, there may or may not be a visible band at the T position (FIG.9).

Example 3

The levels of anti-PLA2R auto-antibodies described herein can bedetermined using an ELISA assay as illustrated in FIG. 10. An ELISAassay comprises performing a standard titration assay and a sample assayin order to determine the amount of anti-PLA2R auto-antibodies in asample obtained from a subject. As shown, the ELISA assay microtiterplate consists of two duplicate reference rows for increasing amounts ofIgG protein. Standard amounts of IgG protein ranging from 0-50 ng/ml orpg/ml are placed in the reference rows to create a standard curve forhuman IgG. Excess amounts of PLA2R are immobilized in the sample wellsof plate. The serum sample is placed in the sample wells. Subsequently,a horse-radish peroxidase labeled anti-human IgG antibody is added tothe wells. The mixtures in the wells are allowed to incubate at roomtemperature for 90 min and the liquid is decanted. The wells are washedfive times with deionized water. Then an aliquot of 3,3′,5,5′tetramethylbenzidine (TMB) reagent is added into each well. The mixtureis gently mixed for 10 seconds and incubated at room temperature (18-25°C.) for 20 minutes. The enzymatic reaction is terminated by adding 1NHCl. Gentle agitation is carried out till all the blue color changes toyellow color completely. The amount of color by-product is determined byreading its absorbance at 450 nm with a microtiter well reader. The A₄₅₀correspond to the amount of human IgG antibodies in the wells. Theamount of the anti-PLA2R auto-antibodies in a test sample can beestimated from the A₄₅₀ obtained from the sample wells and the standardcurve obtained from the reference wells.

In an alternate embodiment, the modified ELISA assay as shown in FIG. 11can be used. As in FIG. 10, the reference rows and sample wells arelabeled (FIG. 11). Excess amounts of PLA2R are immobilized in the wellsof plate. A fixed amount of IgG is placed in duplicate reference wells.This fixed amount is the reference value corresponds to the averageamount of the anti-PLA2R auto-antibodies found in the serum of non-MNhealthy subjects. The sample, serum, is also placed in the duplicatesample wells. The assay plate is process as described herein. The A₄₅₀obtained from the sample wells are compared with those obtained for thecorresponding reference rows in order to determine whether there is anincrease or decrease in the amount of anti-PLA2R auto-antibodies in theserum sample.

TABLE 1 Protein Accession number Size (aa) Chondroitin sulfateproteoglycan 4 NP_001888.2 2322 KIAA0960 NP_056019 1657 M-typephospholipase A2 receptor NP_031392.3 1463 CD 109, Gov system plateletNP_598000.2 1445 alloantigen Crumbs homolog 2 NP_775960.4 1285 NephrinNP_004637.1 1241 Integrin, alpha 1 subunit NP_852478.1 1179 Integrin,alpha 3 subunit NP_002195.1 1051 Membrane alanine aminopeptidaseNP_001141.2 967 Aminopeptidase A NP_001968.3 957 Integrin, beta 1isoform 1D NP_391988.1 801 Neutral endopeptidase NP_009218.2 750Endoglin isoform 2 NP_000109.1 625 Podocalyxin-like isoform 2NP_005388.2 526Partial list of human glomerular proteins identified by massspectrometry based on spectra from peptides common to the approximately200 kDa and 150 kDa regions of the gel. The proteins are arrangedaccording to their predicted size, given in amino acids (aa). Proteinsrepresent both podocyte proteins (nephrin, alpha 3 integrin, neutralendopeptidase) and endothelial proteins (endoglin).

Human PLA2R isoform isoform 2 precursor, NP_001007268 (SEQ. ID. NO. 1) 1mllspsllll lllgaprgca egvaaaltpe rllewqdkgi fviqseslkk ciqagksvlt 61lenckqankh mlwkwvsnhg lfniggsgcl glnfsapeqp lslyecdstl vslrwrcnrk 121mitgplqysv qvandntvva srkyihkwis ygsgggdice ylhkdlhtik gnthgmpcmf 181pfqynhqwhh ectregredd llwcattsry erdekwgfcp dptsaevgcd tiwekdlnsh 241icyqfnllss lswseahssc qmqggtllsi tdeteenfir ehmssktvev wmglnqldeh 301agwqwsdgtp lnylnwspev nfepfvedhc gtfssfmpsa wrsrdcestl pyickkylnh 361idheivekda wkyyathcep gwnpynrncy klqkeektwh ealrscqadn saliditsla 421eveflvtllg denasetwig lssnkipvsf ewsndssvif tnwhtlephi fpnrsqlcvs 481aeqseghwkv knceerlfyi ckkaghvlsd aesgcqegwe rhggfcykid tvlrsfdqas 541sgyycppalv titnrfeqaf itslissvvk mkdsyfwial qdqndtgeyt wkpvgqkpep 601vqythwnthq prysggcvam rgrhplgrwe vkhcrhfkam slckgpvenq ekaeyeerwp 661fhpcyldwes epglascfkv fhsekvlmkr twreaeafce efgahlasfa hieeenfvne 721llhskfnwte erqfwigfnk rnplnagswe wsdrtpvvss fldntyfged arncavykan 781ktllplhcgs krewickipr dvkpkipfwy qydvpwlfyq daeylfhtfa sewlnfefvc 841swlhsdllti hsaheqefih skikalskyg aswwiglqee randefrwrd gtpviyqnwd 901tgrertvnnq sqrcgfissi tglwgseecs vsmpsickrk kvwliekkkd tpkqhgtcpk 961gwlyfnykcl llnipkdpss wknwthaqhf caeeggtlva ieseveqafi tmnlfgqtts 1021vwiglqnddy etwlngkpvv ysnwspfdii nipshnttev qkhiplcall ssnpnfhftg 1081kwyfedcgke gygfvcekmq dtsghgvnts dmypmpntle ygnrtykiin anmtwyaaik 1141tclmhkaqlv sitdqyhqsf ltvvlnrlgy ahwiglfttd nglnfdwsdg tkssftfwkd 1201eessllgdcv fadsngrwhs tacesflqga ichvppetrq sehpelcset sipwikfksn 1261cysfstvlds msfeaahefc kkegsnllti kdeaenafll eelfafgssv qmvwlnaqfd 1321gnskNP_031392.3 Human phospholipase A2 receptor 1 isoform 1 precursor (SEQ.ID. NO. 2) 1mllspsllll lllgaprgca egvaaaltpe rllewqdkgi fviqseslkk ciqagksvlt 61lenckqankh mlwkwvsnhg lfniggsgcl glnfsapeqp lslyecdstl vslrwrcnrk 121mitgplqysv qvandntvva srkyihkwis ygsgggdice ylhkdlhtik gnthgmpcmf 181pfqynhqwhh ectregredd llwcattsry erdekwgfcp dptsaevgcd tiwekdlnsh 241icyqfnllss lswseahssc qmqggtllsi tdeteenfir ehmssktvev wmglnqldeh 301agwqwsdgtp lnylnwspev nfepfvedhc gtfssfmpsa wrsrdcestl pyickkylnh 361idheivekda wkyyathcep gwnpynrncy klqkeektwh ealrscqadn saliditsla 421eveflvtllg denasetwig lssnkipvsf ewsndssvif tnwhtlephi fpnrsqlcvs 481aeqseghwkv knceerlfyi ckkaghvlsd aesgcqegwe rhggfcykid tvlrsfdqas 541sgyycppalv titnrfeqaf itslissvvk mkdsyfwial qdqndtgeyt wkpvgqkpep 601vqythwnthq prysggcvam rgrhplgrwe vkhcrhfkam slckqpvenq ekaeyeerwp 661fhpcyldwes epglascfkv fhsekvlmkr twreaeafce efgahlasfa hieeenfvne 721llhskfnwte erqfwigfnk rnplnagswe wsdrtpvvss fldntyfged arncavykan 781ktllplhcgs krewickipr dvkpkipfwy qydvpwlfyq daeylfhtfa sewlnfefvc 841swlhsdllti hsaheqefih skikalskyg aswwiglqee randefrwrd gtpviyqnwd 901tgrertvnnq sqrcgfissi tglwgseecs vsmpsickrk kvwliekkkd tpkqhgtcpk 961gwlyfnykcl llnipkdpss wknwthaqhf caeeggtlva ieseveqafi tmnlfgqtts 1021vwiglqnddy etwlngkpvv ysnwspfdii nipshnttev qkhiplcall ssnpnfhftg 1081kwyfedcgke gygfvcekmq dtsghgvnts dmypmpntle ygnrtykiin anmtwyaaik 1141tclmhkaqlv sitdqyhqsf ltvvlnrlgy ahwiglfttd nglnfdwsdg tkssftfwkd 1201eessllgdcv fadsngrwhs tacesflqga ichvppetrq sehpelcset sipwikfksn 1261cysfstvlds msfeaahefc kkegsnllti kdeaenafll eelfafgssv qmvwlnaqfd 1321gnnetikwfd gtptdqsnwg irkpdtdyfk phhcvalrip eglwqlspcq ekkgfickme 1381adihtaealp ekgpshsiip lavvltlivi vaictlsfci ykhnggffrr lagfrnpyyp 1441atnfstvyle enilisdlek sdqNM_007366.3 mRNA of the human phospholipase A2 receptor 1 isoform 1precursor (SEQ. ID. NO. 4) 1cccgagtgtc ggttcactgt ggagacagcg gtggcggagt gggtctccag ggctctgggc 61tggcaaggcc cccggagggg gtggggcgcg gaggaggcta cggatccgct tccgcgcggc 121ggggccgggt gcttgggacg cggctctggg ctcccgggat aaggggctcc cgggacaagg 181ggctcccgga gagcccagtg gttagcgatg ctgctgtcgc cgtcgctgct gctgctgctg 241ctgctggggg cgccgcgggg ctgcgccgag ggtgtggcgg cggcgcttac ccccgagcgg 301ctcctggagt ggcaggataa aggaatattt gttatccaaa gtgagagtct caagaaatgc 361attcaagcag gtaaatcggt tctgaccctg gagaactgca agcaagcaaa caagcacatg 421ctgtggaaat gggtttcaaa ccatggcctc tttaacatag gaggcagtgg ttgcctgggc 481ctgaatttct ccgccccaga gcagccatta agcttatatg aatgtgactc caccctcgtt 541tccttacggt ggcgctgtaa caggaagatg atcacaggcc cgctgcagta ctctgtccag 601gtggcgcatg acaacacagt ggtggcctca cggaagtata ttcataagtg gatttcttat 661gggtcaggtg gtggagacat ttgtgaatat ctacacaaag atttgcatac aatcaaaggg 721aacacccacg ggatgccgtg tatgtttccc ttccagtata accatcagtg gcatcatgaa 781tgtacccgtg aaggtcggga agatgactta ctgtggtgtg ccacgacaag ccgttatgaa 841agagatgaaa agtggggatt ttgccctgat cccacctctg cagaagtagg ttgtgatact 901atttgggaga aggacctcaa ttcacacatt tgctaccagt tcaacctgct ttcatctctc 961tcttggagtg aggcacattc ttcatgccag atgcaaggag gtacgctgtt aagtattaca 1021gatgaaactg aagaaaattt cataagggag cacatgagca gtaaaacagt ggaggtgtgg 1081atgggcctca atcagctgga tgaacacgct ggctggcagt ggtctgatgg aacgccgctc 1141aactatctga attggagccc agaggtaaat tttgagccat ttgttgaaga tcactgtgga 1201acatttagtt catttatgcc aagtgcctgg aggagtcggg attgtgagtc caccttgcca 1261tatatatgta aaaaatatct aaaccacatt gatcatgaaa tagttgaaaa agatgcgtgg 1321aaatattatg ctacccactg tgagcctggc tggaatccct acaatcgtaa ttgctacaaa 1381cttcagaaag aagaaaagac ctggcatgag gctctgcgtt cttgtcaggc tgataacagt 1441gcattaatag acataacctc attagcagag gtggagtttc ttgtaaccct ccttggagat 1501gaaaatgcat cagaaacatg gattggtttg agcagcaata aaattccagt ttcctttgaa 1561tggtctaatg actcttcagt catctttact aattggcaca cacttgagcc ccacattttt 1621ccaaatagaa gccagctgtg tgtctcagca gagcagtctg agggacactg gaaagtcaaa 1681aattgtgaag aaagactttt ttacatttgt aaaaaagcag gccatgtcct ctctgatgct 1741gaatcaggat gtcaagaggg atgggagaga catggtggat tctgttacaa aattgacaca 1801gtccttcgaa gctttgacca agcttccagc ggttattact gtcctcctgc acttgtaacc 1861attacaaaca ggtttgaaca ggcttttatt accagtttga tcagtagtgt ggtaaaaatg 1921aaggacagtt atttttggat agctcttcag gaccaaaatg atacgggaga atacacttgg 1981aagccagtag ggcagaaacc cgagccggtg cagtacacac actggaacac acaccagccg 2041cgctacagtg gtggctgtgt tgccatgcga ggaaggcatc cacttggtcg ctgggaagtg 2101aagcactgtc ggcactttaa ggcaatgtcc ttgtgcaagc agccagttga aaatcaggaa 2161aaagcagagt atgaagagag atggcccttt cacccctgct atttggactg ggagtcagag 2221cctggtctgg ccagttgctt caaggtattt catagtgaaa aagttctgat gaaaagaaca 2281tggagagaag ctgaagcatt ttgcgaagaa tttggagctc atcttgcaag ctttgcccat 2341attgaggaag agaattttgt gaatgagctc ttacattcaa aatttaattg gacagaagaa 2401aggcagttct ggattggatt taataaaaga aacccactga atgccggctc atgggagtgg 2461tctgatagaa ctcctgttgt ctcttcgttt ttagacaaca cttattttgg agaagatgca 2521agaaactgtg ctgtttataa ggcaaacaaa acattgctgc ccttacactg tggttccaaa 2581cgtgaatgga tatgcaaaat cccaagagat gtgaaaccca agattccgtt ctggtaccag 2641tacgatgtac cctggctctt ttatcaggat gcagaatacc tttttcatac ctttgcctca 2701gaatggttga actttgagtt tgtctgtagc tggctgcaca gtgatcttct cacaattcat 2761tctgcacatg agcaagaatt catccacagc aaaataaaag cgctatcaaa gtatggtgca 2821agttggtgga ttggacttca agaagaaaga gccaatgatg aatttcgctg gagagatgga 2881acaccagtga tataccagaa ctgggacaca ggaagagaaa gaactgtgaa taatcagagc 2941cagagatgtg gctttatttc ttctataaca ggactctggg gtagtgaaga gtgttcagtt 3001tctatgccta gtatctgtaa gcgaaaaaag gtttggctca tagagaaaaa gaaagataca 3061ccaaaacaac atggaacgtg tcccaaagga tggctatatt ttaactataa gtgccttctg 3121ctgaatatcc ccaaagaccc aagcagttgg aagaactgga cgcatgctca acatttctgt 3181gctgaagaag gggggaccct ggtcgccatt gaaagtgagg tggagcaagc tttcattact 3241atgaatcttt ttggccagac caccagtgtg tggataggtt tacaaaatga tgattatgaa 3301acatggctaa atggaaagcc tgtggtatat tctaactggt ctccatttga tataataaat 3361attccaagtc acaataccac tgaagttcag aaacacattc ctctctgtgc cttactctca 3421agtaatccta attttcattt cactggaaaa tggtattttg aagactgtgg aaaggaaggc 3481tatgggtttg tttgtgaaaa aatgcaagat acttctggac acggtgtaaa tacatctgat 3541atgtatccaa tgcccaatac cttagaatat ggaaacagaa cttacaaaat aattaatgca 3601aatatgactt ggtatgcagc aataaaaacc tgcctgatgc acaaagcaca actggtcagc 3661atcacagacc agtatcacca gtccttcctc actgttgtcc tcaaccggct aggatatgcc 3721cactggattg gactgttcac cacagataat ggtcttaatt ttgactggtc tgatggcacc 3781aaatcttctt tcactttttg gaaagatgag gagtcctccc tccttggtga ctgcgttttt 3841gccgacagca acggacgctg gcatagcaca gcctgcgagt catttctgca aggtgccatt 3901tgtcatgtgc cacctgaaac aagacaatct gaacacccag agttgtgctc agaaacatct 3961attccctgga taaaatttaa aagtaattgc tacagttttt ctacagtcct agacagtatg 4021agttttgagg ctgctcatga attttgcaaa aaggaaggtt ctaatctttt aacaatcaag 4081gatgaggctg aaaatgcatt tctcctagaa gagctgtttg cttttggttc ttctgtccag 4141atggtttggt tgaatgctca atttgatggt aacaatgaaa ccataaagtg gtttgatgga 4201actcccacag accagtcaaa ctggggcatt cggaagccag acacagacta cttcaagccc 4261catcattgtg ttgccttgag gatccctgaa ggattatggc agctatcccc gtgtcaagaa 4321aaaaaaggct ttatatgtaa aatggaggca gatattcaca ctgcagaggc gctgccagaa 4381aaaggaccaa gtcacagcat cattcctctt gcggttgtac tgacactgat agtcattgtg 4441gccatttgca cactttcctt ctgcatatac aagcataacg gtggcttctt caggagactt 4501gcagggtttc ggaatcctta ctatcctgca accaacttta gtacagtata tttagaagaa 4561aatattctca tttctgatct tgagaagagt gaccaataat aatgaggtca gagaatgcca 4621cagacaccag ggtaagtaaa gaagactaaa caggagtctc atctgtcttt ccctttacag 4681cacagatgcc attagaatgt gaattgggtc actattttaa ttattcttga agtgattact 4741ggttttgaat cttaaccaaa tcagatgggt tttgatttat tcatttccct aaactgtgat 4801ccattcttaa aaggggtaaa ttatgcattg gttatttttc agaaagacaa gaactattaa 4861aagaaactcc ctattgNM_001007267 mRNA of Human phospholipase A2 receptor 1 isoform 2 precursor (SEQ. ID. NO. 3) 1cccgagtgtc ggttcactgt ggagacagcg gtggcggagt gggtctccag ggctctgggc 61tggcaaggcc cccggagggg gtggggcgcg gaggaggcta cggatccgct tccgcgcggc 121ggggccgggt gcttgggacg cggctctggg ctcccgggat aaggggctcc cgggacaagg 181ggctcccgga gagcccagtg gttagcgatg ctgctgtcgc cgtcgctgct gctgctgctg 241ctgctggggg cgccgcgggg ctgcgccgag ggtgtggcgg cggcgcttac ccccgagcgg 301ctcctggagt ggcaggataa aggaatattt gttatccaaa gtgagagtct caagaaatgc 361attcaagcag gtaaatcggt tctgaccctg gagaactgca agcaagcaaa caagcacatg 421ctgtggaaat gggtttcaaa ccatggcctc tttaacatag gaggcagtgg ttgcctgggc 481ctgaatttct ccgccccaga gcagccatta agcttatatg aatgtgactc caccctcgtt 541tccttacggt ggcgctgtaa caggaagatg atcacaggcc cgctgcagta ctctgtccag 601gtggcgcatg acaacacagt ggtggcctca cggaagtata ttcataagtg gatttcttat 661gggtcaggtg gtggagacat ttgtgaatat ctacacaaag atttgcatac aatcaaaggg 721aacacccacg ggatgccgtg tatgtttccc ttccagtata accatcagtg gcatcatgaa 781tgtacccgtg aaggtcggga agatgactta ctgtggtgtg ccacgacaag ccgttatgaa 841agagatgaaa agtggggatt ttgccctgat cccacctctg cagaagtagg ttgtgatact 901atttgggaga aggacctcaa ttcacacatt tgctaccagt tcaacctgct ttcatctctc 961tcttggagtg aggcacattc ttcatgccag atgcaaggag gtacgctgtt aagtattaca 1021gatgaaactg aagaaaattt cataagggag cacatgagca gtaaaacagt ggaggtgtgg 1081atgggcctca atcagctgga tgaacacgct ggctggcagt ggtctgatgg aacgccgctc 1141aactatctga attggagccc agaggtaaat tttgagccat ttgttgaaga tcactgtgga 1201acatttagtt catttatgcc aagtgcctgg aggagtcggg attgtgagtc caccttgcca 1261tatatatgta aaaaatatct aaaccacatt gatcatgaaa tagttgaaaa agatgcgtgg 1321aaatattatg ctacccactg tgagcctggc tggaatccct acaatcgtaa ttgctacaaa 1381cttcagaaag aagaaaagac ctggcatgag gctctgcgtt cttgtcaggc tgataacagt 1441gcattaatag acataacctc attagcagag gtggagtttc ttgtaaccct ccttggagat 1501gaaaatgcat cagaaacatg gattggtttg agcagcaata aaattccagt ttcctttgaa 1561tggtctaatg actcttcagt catctttact aattggcaca cacttgagcc ccacattttt 1621ccaaatagaa gccagctgtg tgtctcagca gagcagtctg agggacactg gaaagtcaaa 1681aattgtgaag aaagactttt ttacatttgt aaaaaagcag gccatgtcct ctctgatgct 1741gaatcaggat gtcaagaggg atgggagaga catggtggat tctgttacaa aattgacaca 1801gtccttcgaa gctttgacca agcttccagc ggttattact gtcctcctgc acttgtaacc 1861attacaaaca ggtttgaaca ggcttttatt accagtttga tcagtagtgt ggtaaaaatg 1921aaggacagtt atttttggat agctcttcag gaccaaaatg atacgggaga atacacttgg 1981aagccagtag ggcagaaacc cgagccggtg cagtacacac actggaacac acaccagccg 2041cgctacagtg gtggctgtgt tgccatgcga ggaaggcatc cacttggtcg ctgggaagtg 2101aagcactgtc ggcactttaa ggcaatgtcc ttgtgcaagc agccagttga aaatcaggaa 2161aaagcagagt atgaagagag atggcccttt cacccctgct atttggactg ggagtcagag 2221cctggtctgg ccagttgctt caaggtattt catagtgaaa aagttctgat gaaaagaaca 2281tggagagaag ctgaagcatt ttgcgaagaa tttggagctc atcttgcaag ctttgcccat 2341attgaggaag agaattttgt gaatgagctc ttacattcaa aatttaattg gacagaagaa 2401aggcagttct ggattggatt taataaaaga aacccactga atgccggctc atgggagtgg 2461tctgatagaa ctcctgttgt ctcttcgttt ttagacaaca cttattttgg agaagatgca 2521agaaactgtg ctgtttataa ggcaaacaaa acattgctgc ccttacactg tggttccaaa 2581cgtgaatgga tatgcaaaat cccaagagat gtgaaaccca agattccgtt ctggtaccag 2641tacgatgtac cctggctctt ttatcaggat gcagaatacc tttttcatac ctttgcctca 2701gaatggttga actttgagtt tgtctgtagc tggctgcaca gtgatcttct cacaattcat 2761tctgcacatg agcaagaatt catccacagc aaaataaaag cgctatcaaa gtatggtgca 2821agttggtgga ttggacttca agaagaaaga gccaatgatg aatttcgctg gagagatgga 2881acaccagtga tataccagaa ctgggacaca ggaagagaaa gaactgtgaa taatcagagc 2941cagagatgtg gctttatttc ttctataaca ggactctggg gtagtgaaga gtgttcagtt 3001tctatgccta gtatctgtaa gcgaaaaaag gtttggctca tagagaaaaa gaaagataca 3061ccaaaacaac atggaacgtg tcccaaagga tggctatatt ttaactataa gtgccttctg 3121ctgaatatcc ccaaagaccc aagcagttgg aagaactgga cgcatgctca acatttctgt 3181gctgaagaag gggggaccct ggtcgccatt gaaagtgagg tggagcaagc tttcattact 3241atgaatcttt ttggccagac caccagtgtg tggataggtt tacaaaatga tgattatgaa 3301acatggctaa atggaaagcc tgtggtatat tctaactggt ctccatttga tataataaat 3361attccaagtc acaataccac tgaagttcag aaacacattc ctctctgtgc cttactctca 3421agtaatccta attttcattt cactggaaaa tggtattttg aagactgtgg aaaggaaggc 3481tatgggtttg tttgtgaaaa aatgcaagat acttctggac acggtgtaaa tacatctgat 3541atgtatccaa tgcccaatac cttagaatat ggaaacagaa cttacaaaat aattaatgca 3601aatatgactt ggtatgcagc aataaaaacc tgcctgatgc acaaagcaca actggtcagc 3661atcacagacc agtatcacca gtccttcctc actgttgtcc tcaaccggct aggatatgcc 3721cactggattg gactgttcac cacagataat ggtcttaatt ttgactggtc tgatggcacc 3781aaatcttctt tcactttttg gaaagatgag gagtcctccc tccttggtga ctgcgttttt 3841gccgacagca acggacgctg gcatagcaca gcctgcgagt catttctgca aggtgccatt 3901tgtcatgtgc cacctgaaac aagacaatct gaacacccag agttgtgctc agaaacatct 3961attccctgga taaaatttaa aagtaattgc tacagttttt ctacagtcct agacagtatg 4021agttttgagg ctgctcatga attttgcaaa aaggaaggtt ctaatctttt aacaatcaag 4081gatgaggctg aaaatgcatt tctcctagaa gagctgtttg cttttggttc ttctgtccag 4141atggtttggt tgaatgctca atttgatggt aacagtaagt gatttgggta gaggagagga 4201cataaataaa tacatggttg ttaaagctga tgataatggc atctgtgagc cagaaaactc 4261tccttggata cgttttctga gaaaaaatag catgaagcct aaagccattt cttccaaaaa 4321caacattgca acccttttct ttaccctttt gtcttttaaa ataatcccag aacaccaaaa 4381ataaaaacaa acaataacat gtttatcttt acccttagca ggaagatgct tggctggaac 4441tttgtgttca cagacttagt cattgcatac caaaaccata tttactggaa aatatcccag 4501gtttctaaat gttataaaag cacaatagag ttatggaaat gtttccatga tgaactgtgc 4561tgttaggatt cttatttgct actcataaaa accagagttt gtaataaaat ggaagcatgg 4621tattcttttc tttatgtaat tgatggttat tgaaaggtac ttgtgaagaa aattatttta 4681attggtatgg agagcttgtt acagtggtgt accaaggttg ggggtgagcc taccctttcg 4741aggaggagta ttttaatcac caacattgtt tagaatttca agcagatggt gataataaaa 4801agcagaccaa cttttagtta gctgtattgt tagttttaaa ttattttcag acaatacact 4861attgcccaca catgggatgg actctcctac cgcccctacc cccttggtac atggctggct 4921tggtattaaa gaaattcact gtaaaatctt tttagaaagt gagccatttt gtaatgatga 4981agatgttagg acttcaaagg atttttcttt actcgattag ttttgtttat caaatgattt 5041ctttaaatcg attatatata tatatggaat atttcaaaat tcaaactgtc acattaagaa 5101acatgataac ctaaatgacc taaataagaa cactgtacct aaaataaaga ggcaactttaAmino acid sequence of the extracellular domain of human PLA2R (SEQ.ID. No. 5): 1MLLSPSLLLL LLLGAPRGCA EGVAAALTPE RLLEWQDKGI FVIQSESLKK CIQAGKSVLT 61LENCKQANKH MLWKWVSNHG LFNIGGSGCL GLNFSAPEQP LSLYECDSTL VSLRWRCNRK 121MITGPLQYSV QVAHDNTVVA SRKYIHKWIS YGSGGGDICE YLHKDLHTIK GNTHGMPCMF 181PFQYNHQWHH ECTREGREDD LLWCATTSRY ERDEKWGFCP DPTSAEVGCD TIWEKDLNSH 241ICYQFNLLSS LSWSEAHSSC QMQGGTLLSI TDETEENFIR EHMSSKTVEV WMGLNQLDEH 301AGWQWSDGTP LNYLNWSPEV NFEPFVEDHC GTFSSFMPSA WRSRDCESTL PYICKKYLNH 361IDHEIVEKDA WKYYATHCEP GWNPYNRNCY KLQKEEKTWH EALRSCQADN SALIDITSLA 421EVEFLVTLLG DENASETWIG LSSNKIPVSF EWSNDSSVIF TNWHTLEPHI FPNRSQLCVS 481AEQSEGHWKV KNCEERLFYI CKKAGHVLSD AESGCQEGWE RHGGFCYKID TVLRSFDQAS 541SGYYCPPALV TITNRFEQAF ITSLISSVVK MKDSYFWIAL QDQNDTGEYT WKPVGQKPEP 601VQYTHWNTHQ PRYSGGCVAM RGRHPLGRWE VKHCRHFKAM SLCKQPVENQ EKAEYEERWP 661FHPCYLDWES EPGLASCFKV FHSEKVLMKR TWREAEAFCE EFGAHLASFA HIEEENFVNE 721LLHSKFNWTE ERQFWIGFNK RNPLNAGSWE WSDRTPVVSS FLDNTYFGED ARNCAVYKAN 781KTLLPLHCGS KREWICKIPR DVKPKIPFWY QYDVPWLFYQ DAEYLFHTFA SEWLNFEFVC 841SWLHSDLLTI HSAHEQEFIH SKIKALSKYG ASWWIGLQEE RANDEFRWRD GTPVIYQNWD 901TGRERTVNNQ SQRCGFISSI TGLWGSEECS VSMPSICKRK KVWLIEKKKD TPKQHGTCPK 961GWLYFNYKCL LLNIPKDPSS WKNWTHAQHF CAEEGGTLVA IESEVEQAFI TMNLFGQTTS 1021VWIGLQNDDY ETWLNGKPVV YSNWSPFDII NIPSHNTTEV QKHIPLCALL SSNPNFHFTG 1081KWYFEDCGKE GYGFVCEKMQ DTSGHGVNTS DMYPMPNTLE YGNRTYKIIN ANMTWYAAIK 1141TCLMHKAQLV SITDQYHQSF LTVVLNRLGY AHWIGLFTTD NGLNFDWSDG TKSSFTFWKD 1201EESSLLGDCV FADSNGRWHS TACESFLQGA ICHVPPETRQ SEHPELCSET SIPWIKFKSN 1261CYSFSTVLDS MSFEAAHEFC KKEGSNLLTI KDEAENAFLL EELFAFGSSV QMVWLNAQFD 1321DETIKWFDGT PTDQSNWGIR KPDTDYFKPH HCVALRIPEG LWQLSPCQEK KGFICKMEAD 1381IHTAEALPEK GPSHS

1. (canceled)
 2. (canceled)
 3. An assay comprising measuring forantibodies that are reactive to a phospholipase A2 receptor (PLA2R) in ablood sample obtained from a patient who presents at least one symptomof idiopathic membranous nephropathy (MN) and determining if there is adetectable presence of antibodies reactive against PLA2R wherein thepresence of the detectable level of antibodies indicates that thepatient likely has idiopathic MN.
 4. An assay comprising measuring forantibodies that are reactive to a phospholipase A2 receptor (PLA2R) in ablood sample obtained from a patient who previously has beensuccessfully treated for idiopathic membranous nephropathy (MN) fordetermining the likelihood of relapse of idiopathic MN in the patient,and determining if there is a detectable presence of antibodies reactiveagainst PLA2R wherein a detectable presence of antibodies reactiveagainst PLA2R indicates that the patient likely has a relapse ofidiopathic MN.
 5. An assay comprising: a. measuring a level ofantibodies that are reactive to a phospholipase A2 receptor (PLA2R) in ablood sample obtained from a patient who presents at least one symptomof idiopathic membranous nephropathy (MN), and b. comparing the measuredlevel of the antibodies with a control data from a population of non-MNhealthy individuals, wherein an increase in the antibodies that arereactive to PLA2R in the blood sample over that of the control dataindicates the likelihood of idiopathic MN.
 6. An assay comprising: a.measuring a level of antibodies that are reactive to a phospholipase A2receptor (PLA2R) in a blood sample obtained from a patient whopreviously has been successfully treated for idiopathic membranousnephropathy (MN), and b. comparing the measured level of the antibodieswith a control data from a population of non-MN healthy individuals,wherein an increase in the antibodies that are reactive to PLA2R in theblood sample over that of the control data indicates the likelihood ofrelapse of idiopathic MN.
 7. The assay of claim 3, wherein the PLA2Rantibodies are by measured by light scattering assay or an immunoassay.8. The assay of claim 7, wherein the detectable presence of the PLA2Rantibodies is determined by the detection limit of the light scatteringassay or the immunoassay.
 9. The assay of claim 7, wherein the level ofPLA2R antibodies is measured by the steps comprising: a. contacting theblood sample from the patient with a PLA2R or PLA2R fragment thereof; b.forming an antibody-protein complex between the antibody present in thesample with the PLA2R or PLA2R fragment thereof; c. washing to removeany unbound antibody; d. adding a detection antibody that is labeled andis reactive to the antibody from the sample; e. washing to remove anyunbound labeled detection antibody; and f. converting the label to adetectable signal, wherein the presence of a detectable signal indicatesthe likelihood of idiopathic MN in the patient.
 10. The assay of claim9, wherein the PLA2R or PLA2R protein fragment thereof is deposited orimmobilized on a solid support.
 11. The assay of claim 10, wherein thesupport is in the format of a dipstick, a test strip, a latex bead, amicrosphere or a multi-well plate.
 12. The assay of claim 11, whereinthe detection antibody is labeled by covalently linking to an enzyme,label with a fluorescent compound or metal, or label with achemiluminescent compound.
 13. The assay of claim 3, wherein the symptomis nephrotic syndrome or proteinuria.
 14. The assay of claim 5, whereinthe symptom is nephrotic syndrome or proteinuria.
 15. The assay of claim4, wherein the patient presents a symptom of idiopathic MN.
 16. Theassay of claim 6, wherein the patient presents a symptom of idiopathicMN.
 17. The assay of claim 15, wherein the symptom is nephrotic syndromeor proteinuria.
 18. The assay of claim 16, wherein the symptom isnephrotic syndrome or proteinuria.
 19. The assay of claim 3, furthercomprising selecting the patient for treatment for idiopathic MN withoutsubjecting the patient to a kidney biopsy if there is a detectablepresence of the PLA2R antibodies in the blood sample.
 20. The assay ofclaim 4, further comprising selecting the patient for treatment foridiopathic MN without subjecting the patient to a kidney biopsy if thereis a detectable presence of the PLA2R antibodies in the blood sample.21. The assay of claim 5, further comprising selecting the patient fortreatment for idiopathic MN without subjecting the patient to a kidneybiopsy if there is an increase in PLA2R antibodies in the blood sampleover that of the control data.
 22. The assay of claim 6, furthercomprising selecting the patient for treatment for idiopathic MN withoutsubjecting the patient to a kidney biopsy if there is an increase inPLA2R antibodies in the blood sample over that of the control data.